Oligonucleotides Comprising Modified Nucleosides

ABSTRACT

Polynucleotides, such as aptamers, comprising at least first one 5-position modified pyrimidine and at least one second 5-position modified pyrimidine are provided, wherein the first and second 5-position modified pyrimidines are different. Methods of selecting and using such polynucleotides, such as aptamers, are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/126,330, filed Dec. 18, 2020, which is a division of U.S. applicationSer. No. 16/307,520, filed Dec. 6, 2018, which is a national phase entrypursuant to 35 U.S.C. § 371 of International Application No.PCT/US2017/040299, filed Jun. 30, 2017, which claims the benefit ofpriority of U.S. Provisional Application No. 62/357,623, filed Jul. 1,2016, and U.S. Provisional Application No. 62/437,592, filed Dec. 21,2016, each of which is incorporated by reference herein in its entiretyfor any purpose.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Dec. 30, 2022, isnamed 01137-0020-02US_Sequence_Listing and is 112,410 bytes in size.

FIELD

The present disclosure relates generally to the field ofoligonucleotides comprising modified nucleosides, such as aptamers thatare capable of binding to target molecules. In some embodiments, thepresent disclosure relates to oligonucleotides, such as aptamers, thatcomprise more than one type of base-modified nucleoside, and methods ofmaking and using such aptamers.

BACKGROUND

Modified nucleosides have been used as therapeutic agents, diagnosticagents, and for incorporation into oligonucleotides to improve theirproperties (e.g., stability).

SELEX (Systematic Evolution of Ligands for EXponential Enrichment) is amethod for identifying oligonucleotides (referred to as “aptamers”) thatselectively bind target molecules. The SELEX process is described, forexample, in U.S. Pat. No. 5,270,163. The SELEX method involves theselection and identification of oligonucleotides from a random mixtureof oligonucleotides to achieve virtually any desired criterion ofbinding affinity and selectivity. By introducing specific types ofmodified nucleosides to the oligonucleotides identified in the course ofthe SELEX process, the nuclease stability, net charge, hydrophilicity orlipophilicity may be altered to provide differences in the threedimensional structure and target binding capabilities of theoligonucleotides.

SUMMARY

In some embodiments, an aptamer comprising at least one first 5-positionmodified pyrimidine and at least one second 5-position modifiedpyrimidine is provided, wherein the first 5-position modified pyrimidineand the second 5-position modified pyrimidine are different. In someembodiments, the first 5-position modified pyrimidine is a 5-positionmodified uridine and wherein the second 5-position modified pyrimidineis a 5-position modified cytidine. In some embodiments, the first5-position modified pyrimidine is a 5-position modified cytidine andwherein the second 5-position modified pyrimidine is a 5-positionmodified uridine. In some embodiments, the 5-position modified uridinecomprises a moiety at the 5-position selected from a naphthyl moiety, abenzyl moiety, a tyrosyl moiety, an indole moiety and a morpholinomoiety. In some embodiments, the 5-position modified cytidine comprisesa moiety at the 5-position selected from a naphthyl moiety, a benzylmoiety, a tyrosyl moiety, and a morpholino moiety. In certainembodiments, the moiety is covalently linked to the 5-position of thebase via a linker comprising a group selected from an amide linker, acarbonyl linker, a propynyl linker, an alkyne linker, an ester linker, aurea linker, a carbamate linker, a guanidine linker, an amidine linker,a sulfoxide linker, and a sulfone linker. In some embodiments, the5-position modified cytidine is selected from NapdC, 2NapdC, TyrdC, andPPdC. In some embodiments, the 5-position modified uridine is selectedfrom NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU. In someembodiments, the at least one first 5-position modified pyrimidine is aNapdC and the at least one second 5-position modified pyrimidine isselected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, and ThrdU. In someembodiments, the at least one first 5-position modified pyrimidine is aPPdC and the at least one second 5-position modified pyrimidine isselected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU. Insome embodiments, the at least one second 5-position modified pyrimidineis a TyrdU. In some embodiments, the first 5-position modifiedpyrimidine and the second 5-position modified pyrimidine are capable ofbeing incorporated by a polymerase enzyme. In some embodiments, thefirst 5-position modified pyrimidine and the second 5-position modifiedpyrimidine are capable of being incorporated by a KOD DNA polymerase.

In some embodiments, the aptamer binds a target protein selected fromPCSK9, PSMA, ErbB1, ErbB2, FXN, KDM2A, IGF1R, pIGF1R, a1-Antritrypsin,CD99, MMP28 and PPM.

In some embodiments, the aptamer comprises a region at the 5′ end of theaptamer that is at least 10, at least 15, at least 20, at least 25 or atleast 30 nucleotides in length, or 5 to 30, 10 to 30, 15 to 30, 5 to 20,or 10 to 20 nucleotides in length, wherein the region at the 5′ end ofthe aptamer lacks 5-position modified pyrimidines. In some embodiments,the aptamer comprises a region at the 3′ end of the aptamer that is atleast 10, at least 15, at least 20, at least 25 or at least 30nucleotides in length, or 5 to 30, 10 to 30, 15 to 30, 5 to 20, or 10 to20 nucleotides in length, wherein the region at the 3′ end of theaptamer lacks 5-position modified pyrimidines. In some embodiments, theaptamer is 20 to 100, or 20 to 90, or 20 to 80, or 20 to 70, or 20 to60, or 20 to 50, or 30 to 100, or 30 to 90, or 30 to 80, or 30 to 70, or30 to 60, or 30 to 50, or 40 to 100, or 40 to 90, or 40 to 80, or 40 to70, or 40 to 60, or 40 to 50 nucleotides in length.

In some embodiments, the aptamer has improved nuclease stabilitycompared to an aptamer of the same length and nucleobase sequence thatcomprises an unmodified pyrimidine in place of each of the first5-position modified pyrimidines and/or an unmodified pyrimidine in placeof each of the second 5-position modified pyrimidine. In someembodiments, the aptamer has a longer half-life in human serum comparedto an aptamer of the same length and nucleobase sequence that comprisesan unmodified pyrimidine in place of each of the first 5-positionmodified pyrimidines or an unmodified pyrimidine in place of each of thesecond 5-position modified pyrimidine.

In some embodiments, a composition comprising a plurality ofpolynucleotides is provided, wherein each polynucleotide comprises atleast one first 5-position modified pyrimidine and at least one second5-position modified pyrimidine, wherein the first 5-position modifiedpyrimidine and the second 5-position modified pyrimidine are different.In some embodiments, each polynucleotide comprises a fixed region at the5′ end of the polynucleotide. In some embodiments, the fixed region atthe 5′ end of each polynucleotide is at least 10, at least 15, at least20, at least 25 or at least 30 nucleotides in length, or 5 to 30, 10 to30, 15 to 30, 5 to 20, or 10 to 20 nucleotides in length. In someembodiments, each polynucleotide comprises a fixed region at the 3′ endof the polynucleotide. In some embodiments, the fixed region at the 3′end of the polynucleotide is at least 10, at least 15, at least 20, atleast 25 or at least 30 nucleotides in length, or 5 to 30, 10 to 30, 15to 30, 5 to 20, or 10 to 20 nucleotides in length. In some embodiments,the first 5-position modified pyrimidine is a 5-position modifieduridine and wherein the second 5-position modified pyrimidine is a5-position modified cytidine. In some embodiments, the first 5-positionmodified pyrimidine is a 5-position modified cytidine and wherein thesecond 5-position modified pyrimidine is a 5-position modified uridine.In some embodiments, the 5-position modified uridine comprises a moietyat the 5-position selected from a naphthyl moiety, a benzyl moiety, atyrosyl moiety, a tryptophanyl moiety, an indole moiety and a morpholinomoiety. In some embodiments, the 5-position modified cytidine comprisesa moiety at the 5-position selected from a naphthyl moiety, a benzylmoiety, a tyrosyl moiety and a morpholino moiety. In certainembodiments, the moiety is covalently linked to the 5-position of thebase via a linker comprising a group selected from an amide linker, acarbonyl linker, a propynyl linker, an alkyne linker, an ester linker, aurea linker, a carbamate linker, a guanidine linker, an amidine linker,a sulfoxide linker, and a sulfone linker. In some embodiments, the5-position modified cytidine is selected from NapdC, 2NapdC, TyrdC, andPPdC. In some embodiments, the 5-position modified uridine is selectedfrom NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU. In some embodiments,the at least one first 5-position modified pyrimidine is a NapdC and theat least one second 5-position modified pyrimidine is selected fromNapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU. In someembodiments, the at least one first 5-position modified pyrimidine is aPPdC and the at least one second 5-position modified pyrimidine isselected from NapdU, 2NapdU, PPdU, MOEdU, TrydU, TrpdU, and ThrdU. Insome embodiments, the at least one second 5-position modified pyrimidineis a TyrdU. In some embodiments, the first 5-position modifiedpyrimidine and the second 5-position modified pyrimidine are capable ofbeing incorporated by a polymerase enzyme. In some embodiments, thefirst 5-position modified pyrimidine and the second 5-position modifiedpyrimidine are capable of being incorporated by a KOD DNA polymerase.

In some embodiments, each polynucleotide of the composition comprises arandom region. In some embodiments, the random region is 20 to 100, or20 to 90, or 20 to 80, or 20 to 70, or 20 to 60, or 20 to 50, or 20 to40, or 30 to 100, or 30 to 90, or 30 to 70, or 30 to 60, or 30 to 50, or30 to 40 nucleotides in length. In some embodiments, each polynucleotideis 20 to 100, or 20 to 90, or 20 to 80, or 20 to 70, or 20 to 60, or 20to 50, or 30 to 100, or 30 to 90, or 30 to 80, or 30 to 70, or 30 to 60,or 30 to 50, or 40 to 100, or 40 to 90, or 40 to 80, or 40 to 70, or 40to 60, or 40 to 50 nucleotides in length.

In some embodiments, a composition is provided, comprising a firstaptamer, a second aptamer, and a target, wherein the first aptamercomprises at least one first 5-position modified pyrimidine and at leastone second 5-position modified pyrimidine; wherein the second aptamercomprises at least one third 5-position modified pyrimidine or whereinthe second aptamer comprises at least one third 5-position modifiedpyrimidine and at least one fourth 5-position modified pyrimidine;wherein the first aptamer, second aptamer and the target are capable offorming a trimer complex; and wherein the first 5-position modifiedpyrimidine and the second 5-position modified pyrimidine are different5-position modified pyrimidines.

In some embodiments, the first 5-position modified pyrimidine is a5-position modified uridine and wherein the second 5-position modifiedpyrimidine is a 5-position modified cytidine. In some embodiments, thefirst 5-position modified pyrimidine is a 5-position modified cytidineand wherein the second 5-position modified pyrimidine is a 5-positionmodified uridine. In some embodiments, the 5-position modified uridinecomprises a moiety at the 5-position selected from a naphthyl moiety, abenzyl moiety, a tyrosyl moiety, an indole moiety and a morpholinomoiety. In some embodiments, the 5-position modified cytidine comprisesa moiety at the 5-position selected from a naphthyl moiety, a benzylmoiety, a tyrosyl moiety and a morpholino moiety. In certainembodiments, the moiety is covalently linked to the 5-position of thebase via a linker comprising a group selected from an amide linker, acarbonyl linker, a propynyl linker, an alkyne linker, an ester linker, aurea linker, a carbamate linker, a guanidine linker, an amidine linker,a sulfoxide linker, and a sulfone linker. In some embodiments, the5-position modified cytidine is selected from NapdC, 2NapdC, TyrdC, andPPdC. In some embodiments, the 5-position modified uridine is selectedfrom NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU. In someembodiments, the at least one first 5-position modified pyrimidine is aNapdC and the at least one second 5-position modified pyrimidine isselected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU. Insome embodiments, the at least one first 5-position modified pyrimidineis a PPdC and the at least one second 5-position modified pyrimidine isselected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU. Insome embodiments, the at least one second 5-position modified pyrimidineis a TyrdU. In some embodiments, the first 5-position modifiedpyrimidine and the second 5-position modified pyrimidine are capable ofbeing incorporated by a polymerase enzyme. In some embodiments, thefirst 5-position modified pyrimidine and the second 5-position modifiedpyrimidine are capable of being incorporated by a KOD DNA polymerase.

In some embodiments, the third 5-position modified pyrimidine isselected from a 5-position modified cytidine and a 5-position modifiedpyrimidine. In some embodiments, the third 5-position modifiedpyrimidine and the fourth 5-position modified pyrimidine are different5-position modified pyrimidines. In some embodiments, third 5-positionmodified pyrimidine is a 5-position modified cytidine and the fourth5-position modified pyrimidine is a 5-position modified uridine. In someembodiments, the third 5-position modified cytidine is selected fromBndC, PEdC, PPdC, NapdC, 2NapdC, NEdC, 2NEdC, and TyrdC. In someembodiments, the 5-position modified uridine is selected from BNdU,NapdU, PEdU, IbdU, FBndU, 2NapdU, NEdU, MBndU, BFdU, BTdU, PPdU, MOEdU,TyrdU, TrpdU, and ThrdU.

In some embodiments, the target is selected from a protein, a peptide, acarbohydrate, a small molecule, a cell and a tissue.

In some embodiments, a method is provided, comprising:

-   -   (a) contacting an aptamer capable of binding to a target        molecule with a sample;    -   (b) incubating the aptamer with the sample to allow an        aptamer-target complex to form;    -   (c) enriching for the aptamer-target complex in the sample and    -   (c) detecting for the presence of the aptamer, aptamer-target        complex or target molecule, wherein the detection of the        aptamer, aptamer-target complex or target molecule indicates        that the target molecule is present in the sample, and wherein        the lack of detection of the aptamer, aptamer-target complex or        target molecule indicates that the target molecule is not        present in the sample;    -   wherein the aptamer is a dual-modified aptamer provided herein.        In some embodiments, the method comprises at least one        additional step selected from: adding a competitor molecule to        the sample; capturing the aptamer-target complex on a solid        support; and adding a competitor molecule and diluting the        sample; wherein the at least one additional step occurs after        step (a) or step (b). In some embodiments, the competitor        molecule is selected from a polyanionic competitor. In some        embodiments, the polyanionic competitor is selected from an        oligonucleotide, polydextran, DNA, heparin and dNTPs. In some        embodiments, polydextran is dextran sulfate; and DNA is herring        sperm DNA or salmon sperm DNA. In some embodiments, the target        molecule is selected from a protein, a peptide, a carbohydrate,        a small molecule, a cell and a tissue. In some embodiments, the        sample is selected from whole blood, leukocytes, peripheral        blood mononuclear cells, plasma, serum, sputum, breath, urine,        semen, saliva, meningial fluid, amniotic fluid, glandular fluid,        lymph fluid, nipple aspirate, bronchial aspirate, synovial        fluid, joint aspirate, cells, a cellular extract, stool, tissue,        a tissue biopsy, and cerebrospinal fluid.

In some embodiments, a method for detecting a target in a sample isprovided, comprising

-   -   a) contacting the sample with a first aptamer to form a mixture,        wherein the first aptamer is capable of binding to the target to        form a first complex;    -   b) incubating the mixture under conditions that allow for the        first complex to form;    -   c) contacting the mixture with a second aptamer, wherein the        second aptamer is capable of binding the first complex to form a        second complex;    -   d) incubating the mixture under conditions that allow for the        second complex to form;    -   e) detecting for the presence or absence of the first aptamer,        the second aptamer, the target, the first complex or the second        complex in the mixture, wherein the presence of the first        aptamer, the second aptamer, the target, the first complex or        the second complex indicates that the target is present in the        sample;    -   wherein the first aptamer comprises at least one first        5-position modified pyrimidine and at least one second        5-position modified pyrimidine;    -   wherein the second aptamer comprises at least one third        5-position modified pyrimidine, or wherein the second aptamer        comprises at least one third 5-position modified pyrimidine and        at least one fourth 5-position modified pyrimidine;    -   wherein the first 5-position modified pyrimidine and the second        5-position modified pyrimidine are different 5-position modified        pyrimidines.

In some embodiments, the first 5-position modified pyrimidine is a5-position modified uridine and wherein the second 5-position modifiedpyrimidine is a 5-position modified cytidine. In some embodiments, thefirst 5-position modified pyrimidine is a 5-position modified cytidineand wherein the second 5-position modified pyrimidine is a 5-positionmodified uridine. In some embodiments, the 5-position modified uridinecomprises a moiety at the 5-position selected from a naphthyl moiety, abenzyl moiety, a tyrosyl moiety, an indole moiety and a morpholinomoiety. In some embodiments, the 5-position modified cytidine comprisesa moiety at the 5-position selected from a naphthyl moiety, a benzylmoiety, a tyrosyl moiety and a morpholino moiety. In certainembodiments, the moiety is covalently linked to the 5-position of thebase via a linker comprising a group selected from an amide linker, acarbonyl linker, a propynyl linker, an alkyne linker, an ester linker, aurea linker, a carbamate linker, a guanidine linker, an amidine linker,a sulfoxide linker, and a sulfone linker. In some embodiments, the5-position modified cytidine is selected from NapdC, 2NapdC, TyrdC, andPPdC. In some embodiments, the 5-position modified uridine is selectedfrom NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU. In someembodiments, the at least one first 5-position modified pyrimidine is aNapdC and the at least one second 5-position modified pyrimidine isselected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, and ThrdU. In someembodiments, the at least one first 5-position modified pyrimidine is aPPdC and the at least one second 5-position modified pyrimidine isselected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU. Insome embodiments, the at least one second 5-position modified pyrimidineis a TyrdU. In some embodiments, the first 5-position modifiedpyrimidine and the second 5-position modified pyrimidine are capable ofbeing incorporated by a polymerase enzyme. In some embodiments, thefirst 5-position modified pyrimidine and the second 5-position modifiedpyrimidine are capable of being incorporated by a KOD DNA polymerase.

In some embodiments, the third 5-position modified pyrimidine isselected from a 5-position modified cytidine and a 5-position modifiedpyrimidine. In some embodiments, the third 5-position modifiedpyrimidine and the fourth 5-position modified pyrimidine are different5-position modified pyrimidines. In some embodiments, third 5-positionmodified pyrimidine is a 5-position modified cytidine and the fourth5-position modified pyrimidine is a 5-position modified uridine. In someembodiments, the third 5-position modified cytidine is selected fromBndC, PEdC, PPdC, NapdC, 2NapdC, NEdC, 2NEdC, and TyrdC. In someembodiments, the 5-position modified uridine is selected from BNdU,NapdU, PedU, IbdU, FbndU, 2NapdU, NedU, MbndU, BfdU, BtdU, PpdU, MOEdU,TyrdU, TrpdU, and ThrdU.

In some embodiments, the target molecule is selected from a protein, apeptide, a carbohydrate, a small molecule, a cell and a tissue. In someembodiments, the first aptamer, second aptamer and the target arecapable of forming a trimer complex.

In some embodiments, a method for identifying one or more aptamerscapable of binding to a target molecule is provided, comprising:

-   -   (a) contacting a library of aptamers with the target molecule to        form a mixture, and allowing for the formation of an        aptamer-target complex, wherein the aptamer-target complex forms        when an aptamer has affinity for the target molecule;    -   (b) partitioning the aptamer-target complex from the remainder        of the mixture (or enriching for the aptamer-target complex);    -   (c) dissociating the aptamer-target complex; and    -   (d) identifying the one or more aptamers capable of binding to        the target molecule;    -   wherein the library of aptamers comprises a plurality of        polynucleotides, wherein each polynucleotide comprises at least        one first 5-position modified pyrimidine and at least one second        5-position modified pyrimidine, wherein the first 5-position        modified pyrimidine and the second 5-position modified        pyrimidine are different 5-position modified pyrimidines. In        some embodiments, steps (a), (b) and/or (c) are repeated at        least one time, two times, three times, four times, five times,        six times, seven times, eight times, nine times or ten times.

In some embodiments, the one or more aptamers capable of binding to thetarget molecule are amplified. In some embodiments, the mixturecomprises a polyanionic competitor molecule. In some embodiments, thepolyanionic competitor is selected from an oligonucleotide, polydextran,DNA, heparin and dNTPs. In some embodiments, polydextran is dextransulfate; and DNA is herring sperm DNA or salmon sperm DNA.

In some embodiments, the target molecule is selected from a protein, apeptide, a carbohydrate, a small molecule, a cell and a tissue.

In some embodiments, each polynucleotide comprises a fixed region at the5′ end of the polynucleotide. In some embodiments, the fixed region atthe 5′ end of each polynucleotide is at least 10, at least 15, at least20, at least 25 or at least 30 nucleotides in length, or 5 to 30, 10 to30, 15 to 30, 5 to 20, or 10 to 20 nucleotides in length. In someembodiments, each polynucleotide comprises a fixed region at the 3′ endof the polynucleotide. In some embodiments, the fixed region at the 3′end of the polynucleotide is at least 10, at least 15, at least 20, atleast 25 or at least 30 nucleotides in length, or 5 to 30, 10 to 30, 15to 30, 5 to 20, or 10 to 20 nucleotides in length.

In some embodiments, the first 5-position modified pyrimidine is a5-position modified uridine and wherein the second 5-position modifiedpyrimidine is a 5-position modified cytidine. In some embodiments, thefirst 5-position modified pyrimidine is a 5-position modified cytidineand wherein the second 5-position modified pyrimidine is a 5-positionmodified uridine. In some embodiments, the 5-position modified uridinecomprises a moiety at the 5-position selected from a naphthyl moiety, abenzyl moiety, a tyrosyl moiety, an indole moiety and a morpholinomoiety. In some embodiments, the 5-position modified cytidine comprisesa moiety at the 5-position selected from a naphthyl moiety, a benzylmoiety, a tyrosyl moiety and a morpholino moiety. In certainembodiments, the moiety is covalently linked to the 5-position of thebase via a linker comprising a group selected from an amide linker, acarbonyl linker, a propynyl linker, an alkyne linker, an ester linker, aurea linker, a carbamate linker, a guanidine linker, an amidine linker,a sulfoxide linker, and a sulfone linker. In some embodiments, the5-position modified cytidine is selected from NapdC, 2NapdC, TyrdC, andPPdC. In some embodiments, the 5-position modified uridine is selectedfrom NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU. In some embodiments,the at least one first 5-position modified pyrimidine is a NapdC and theat least one second 5-position modified pyrimidine is selected fromNapdU, 2NapdU, PPdU, MOEdU, TrydU, TrpdU, and ThrdU. In someembodiments, the at least one first 5-position modified pyrimidine is aPPdC and the at least one second 5-position modified pyrimidine isselected from NapdU, 2NapdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU. Insome embodiments, the at least one second 5-position modified pyrimidineis a TyrdU. In some embodiments, the first 5-position modifiedpyrimidine and the second 5-position modified pyrimidine are capable ofbeing incorporated by a polymerase enzyme. In some embodiments, thefirst 5-position modified pyrimidine and the second 5-position modifiedpyrimidine are capable of being incorporated by a KOD DNA polymerase.

In some embodiments, each polynucleotide comprises a random region. Insome embodiments, the random region is 20 to 100, or 20 to 90, or 20 to80, or 20 to 70, or 20 to 60, or 20 to 50, or 20 to 40, or 30 to 100, or30 to 90, or 30 to 70, or 30 to 60, or 30 to 50, or 30 to 40 nucleotidesin length. In some embodiments, each polynucleotide is 20 to 100, or 20to 90, or 20 to 80, or 20 to 70, or 20 to 60, or 20 to 50, or 30 to 100,or 30 to 90, or 30 to 80, or 30 to 70, or 30 to 60, or 30 to 50, or 40to 100, or 40 to 90, or 40 to 80, or 40 to 70, or 40 to 60, or 40 to 50nucleotides in length.

In some embodiments, each polynucleotide is an aptamer that binds atarget, and wherein the library comprises at least 1000 aptamers,wherein each aptamer comprises a different nucleotide sequence.

In some embodiments, an aptamer that binds PCSK9 protein is provided. Insome such embodiments, the aptamer comprises the sequence 5′-yGpppG-3′,wherein each y is a TyrdU and each p is a NapdC. In some embodiments,the aptamer further comprises the sequence 5′-yEAyGA_(n)pAp-3′, whereinE is selected from y, A, and G; and n is 0 or 1. In some embodiments, nis 0. In some embodiments, the sequence 5′-yEAyGA_(n)pAp-3′ is located5′ of the sequence 5′-yGpppG-3′. In some embodiments, E is y.

In some embodiments, an aptamer that binds PCSK9 is provided, whereinthe aptamer comprises the sequence 5′-F_(n)pppAAGRJrpRppW_(m)-3′ (SEQ IDNO: 81), wherein F is selected from r and G; each R is independentlyselected from G and A; J is selected from r and A; W is selected from r,G, and A; n is 0 or 1; m is 0 or 1; r is PpdC; and p is NapdU. In someembodiments, m is 1. In some embodiments, F is r. In some embodiments, Jis r. In some embodiments, W is G.

In some embodiments, an aptamer that binds PCSK9 is provided, whereinthe aptamer comprises the sequence 5′-TTppGGpp-3′, wherein each p is aNapdC.

In some embodiments, an aptamer that binds PCSK9 is 20 to 100, or 20 to90, or 20 to 80, or 20 to 70, or 20 to 60, or 20 to 50, or 30 to 100, or30 to 90, or 30 to 80, or 30 to 70, or 30 to 60, or 30 to 50, or 40 to100, or 40 to 90, or 40 to 80, or 40 to 70, or 40 to 60, or 40 to 50nucleotides in length.

In some embodiments, the aptamer inhibits PCSK9 binding to LDL-R. Insome embodiments, the aptamer inhibits PCSK9 binding to LDL-R with anIC₅₀ of less than 30 nM, less than 20 nM, or less than 15 nM.

In some embodiments, a method of lowering cholesterol in a subject isprovided, comprising administering to a subject in need thereof anaptamer that binds PCSK9. In some embodiments, the aptamer that bindsPCSK9 is an aptamer provided herein. In some embodiments, thecholesterol is low-density lipoprotein (LDL) cholesterol (LDL-C). Insome embodiments, the subject has heterozygous familialhypercholesterolemia or clinical atherosclerotic cardiovascular disease(CVD).

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Selection of nucleic acid aptamers modified with DNA librariescontaining C5-position modified uridine and cytidine triphosphates.Schematics of selection with two modified bases. Outline of selectionmethod in which 30N randomized chemically synthesized master antisensebiotinylated template library was used to enzymatically synthesizevarious modified and unmodified libraries by primer extension reactions.

FIG. 2 . Binding affinities of 40mer (30N+5+5) aptamers to PCSK9generated using various modified libraries. Aptamers with affinities ≥1nM are highlighted in gray shade and aptamers shown at 320 nM affinityrepresent no detectable binding at 32 nM top concentration on bindingcurve. Black line on each of the library indicates median value for theall aptamers in that library.

FIG. 3 . The affinity and percent copy number for single modified,either dU or dC vs two modified dC with dU. Each dot represents one ofthe aptamers with affinity values shown on Y-axis and their percent copynumber on X-axis. Red dots are single modified aptamers and green dots(open and filled) are two modified aptamers. Filled green dots representsome Nap-dC/Tyr-dU and PP-dC/Tyr-dU aptamers.

FIG. 4A-B. Truncability of single base modified and two base modifiedaptamers. (A) All high affinity 40mer sequences were further truncatedto 30mer to their random region length only removing 5 nucleotides fromeach of the 5′ and 3′ ends. The percentage of aptamers that retain orhave improved binding affinity to PCSK9 is plotted on Y-axis. (B)Affinity comparisons and truncability of single base modified and twobase modified aptamers from each individual library. Aptamers withaffinities ≥1 nM are highlighted in gray shade, the highest averageaffinities were for aptamers with two modified base combinations ofPP-dC with PP-dU, Nap-dU and Tyr-dU.

FIG. 5 . Target binding specificity of three high affinity aptamers fromeach library to other proprotein convertases (PCs). The solutionaffinity measurements were carried out for thirty-three aptamers(40mers) total, with eleven aptamers having a single modified base(i.e., three aptamers having Nap-dC/dT; three aptamers having dC/Nap-dU;three aptamers having dC/Pp-dU and two aptamers having dC/Ty-dU) andtwenty-two aptamers having double modified base (i.e., three aptamershaving Nap-dC/Nap-dU; three aptamers having Nap-dC/Pp-dU; three aptamershaving Nap-dC/Moe-dU; three aptamers having Nap-dC/Tyr-dU; threeaptamers having Pp-dC/Pp-dU; three aptamers having Pp-dC/Nap-dU; threeaptamers having Pp-Ty-dU, and one aptamer having Pp-dC/Moe-dU). Theaptamers below dotted line at 100 nM affinity indicates no detectablebinding at 100 nM concentration. The affinities to remaining PCs (PCSK5,PCSK6 and PCSK8) were not tested.

FIG. 6 . Species cross-reactivity of single base and two base modifiedaptamers. Affinity of single modified (three aptamers) and two modified(38 aptamers) truncated 30-mer aptamers (K_(d) value ≤1 nM) to PCSK9from human, monkey, mouse and rat. The single modified aptamers bound tothe human and monkey PSCKS9, but not to the mouse or rate PSKC9. Incontrast, the two modified aptamers bound to human, monkey, mouse andrat. The percent identity of the PCSK9 protein from each species isprovided relative to the human PSCK9.

FIG. 7A-C. Sandwich pair screening in bead-based Luminex® assay. (A)Schematics of aptamer sandwich pair screening. (B) Sandwich pairsshowing signal of greater than or equal to 50-fold at 10 nM PCSK9concentration compared with no protein in buffer. All the aptamerstested in the sandwich assay were 40mers having a Kd≤1 nM. A total of 70pairs showed signals of ≥50-fold. Three sandwich pairs were identifiedwhen each aptamer of the pair were selected from single modifiedlibraries (3 sandwich aptamer pairs/3 single base modified libraries).In contrast, 22 sandwich pairs were identified when one aptamer of thepair was selected from three single base modified libraries and theother aptamer of the pairs was selected from four double base modifiedlibraries (i.e., 22 sandwich aptamer pairs/3 single base modifiedlibraries, and 4 double base modified libraries), and 45 sandwich pairswere identified when both aptamers of the pairs were selected from dualmodified libraries (45 sandwich aptamer pairs/5 double base modifiedlibraries). (C) Comparison of the number of sandwich pairs for thetarget protein PCSK9 derived from the capture aptamer library having asingle base modified aptamer and the detection aptamer library having asingle base modified aptamer; the capture aptamer library having asingle base modified aptamer and the detection aptamer library havingtwo base modified aptamer; and the capture aptamer library having twobase modified aptamer and the detection aptamer library having two basemodified aptamer.

FIG. 8A-D. Sandwich pairs showing PCSK9 concentration dependent signalsin bead-based Luminex® assays. (A) The concentration dependent signalswere observed with best performing capture or primary aptamer pairedwith select secondary or detection aptamers. (B) The concentrationdependent signals were observed with best performing secondary ordetection aptamer with select primary or capture aptamers. (C) Leadsandwich pair, dC/PP-dU aptamer (primary) and Nap-dC/Nap-dU aptamer(secondary), showing signals when orientation of the aptamers isswitched. (D) The standard curve obtained with recombinant wild typePCSK9 and the gain-of-function mutant PCSK9 D374Y. The linearconcentration dependent signals were obtained with sandwich pairdetecting wild-type PCSK9 (circles) and the gain-of-function mutantPCSK9 D374Y (triangles) protein.

FIG. 9A-B. Sensitivity of sandwich assay: performance of aptamersandwich assay (dC/PP-dU aptamer (primary) and Nap-dC/Nap-dU aptamer(secondary)) showing limits of detection of PCSK9 concentrations inbuffer. (A) The linear concentration dependent signals were observedwith aptamer sandwich assay with lower limit of quantification ˜80 pg/mL(LLOQ) (B) The linear concentration dependent signals were observed withaptamer sandwich assay with upper limit of quantification ˜10 ng/mL(ULOQ).

FIG. 10 . Dilution linearity of the sandwich assay using dC/PP-dUaptamer (primary) and Nap-dC/Nap-dU aptamer (secondary).

FIG. 11 . The sandwich assay comprising primary single base modifiedaptamer and secondary two base modified aptamer (dC/PP-dU aptamer(primary) and Nap-dC/Nap-dU aptamer (secondary)).

FIG. 12 . Over expression of PCSK9 in wild-type HepG2 cells.

FIG. 13 . Plate-based in vitro PCSK9 inhibition assays: schematics ofinhibition of PCSK9 with aptamers.

FIG. 14 . Inhibition screen for single base or two base modifiedaptamers.

FIG. 15A-B. Species cross-reactive potential therapeutic two basemodified aptamers. (A) The 30mer two base modified (PP-dC/Nap-dU) rodentcross-reactive aptamer (11733-44, SEQ ID No: 44) affinities to humanPCSK9 (filled blue circle), human gain-of-function mutant PCSK9 D374Y(filled red circle), Rhesus monkey PCSK9 (filled green square), ratPCSK9 (filled pink hexagon), mouse PCSK9 (filled inverted triangle) andscrambled control aptamer (open black diamond). (B) Speciescross-reactive potential therapeutic two base modified aptamer showinginhibition of PCSK9 interaction with LDL-R. aptamer potently inhibitingPCSK9 interaction with LDL-R at EC₅₀ value of 2.1 nM (blue filledcircle) and PCSK9 D374Y at EC₅₀ value of 3.6 nM (red filled triangle)and the scrambled control aptamer showing no inhibition of wild typePCSK9 (green filled squares) and gain-of-function mutant PCSK9 D374Y(open black squares).

FIG. 16 . Schematics of LDL-uptake reversal assay.

FIG. 17 . Species cross-reactive PP-dC/Nap-DU aptamer inhibited LDL-Rdegradation by blocking PCSK9 interaction with LDL-R and increases LDL-Rlevels on the surface of HepG2 cells. The EC₅₀ value for the LDL-uptakereversal is 13.5 nM by active SOMAmer (red circle) which was notobserved with scrambled control of the same sequence (blue triangle).

FIG. 18 . Stability of single modified and dual modified aptamers in 90%human serum over time. The modification pattern of single C-5 modifiedor dual C-5 modified aptamers is provided with the figure legend (e.g.,X/Y where X represents a dC (non-modified nucleotide), NapdC (Nap), orPPdC (PP); and Y represents a dU or dT (non-modified nucleotide, TyrdU(Tyr), NapdU (Nap), PPdU (PP) or MOEdU (MOE)).

FIG. 19A-C. Binding affinities of 40mer (30N+5+5) aptamers to ErbB2 (A),ErbB3 (B), and PSMA (C) generated using various modified libraries.Aptamers with affinities ≥1 nM are highlighted in gray shade andaptamers shown at 320 nM affinity represent no detectable binding at 32nM top concentration on binding curve. Black line on each of the libraryindicates median value for the all aptamers in that library.

FIG. 20 . Certain exemplary 5-position modified uridines and cytidinesthat may be incorporated into aptamers.

FIG. 21 . Certain exemplary modifications that may be present at the5-position of uridine. The chemical structure of the C-5 modificationincludes the exemplary amide linkage that links the modification to the5-position of the uridine. The 5-position moieties shown include abenzyl moiety (e.g., Bn, PE and a PP), a naphthyl moiety (e.g., Nap,2Nap, NE), a butyl moiety (e.g, iBu), a fluorobenzyl moiety (e.g., FBn),a tyrosyl moiety (e.g., a Tyr), a 3,4-methylenedioxy benzyl (e.g., MBn),a morpholino moiety (e.g., MOE), a benzofuranyl moiety (e.g., BF), anindole moiety (e.g, Trp) and a hydroxypropyl moiety (e.g., Thr).

FIG. 22 . Certain exemplary modifications that may be present at the5-position of cytidine. The chemical structure of the C-5 modificationincludes the exemplary amide linkage that links the modification to the5-position of the cytidine. The 5-position moieties shown include abenzyl moiety (e.g., Bn, PE and a PP), a naphthyl moiety (e.g., Nap,2Nap, NE, and 2NE) and a tyrosyl moiety (e.g., a Tyr).

DETAILED DESCRIPTION

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. “Comprising A or B” means including A, or B, or Aand B. It is further to be understood that all base sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription.

Further, ranges provided herein are understood to be shorthand for allof the values within the range. For example, a range of 1 to 50 isunderstood to include any number, combination of numbers, or sub-rangefrom the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50 (as well as fractions thereof unless the context clearly dictatesotherwise). Any concentration range, percentage range, ratio range, orinteger range is to be understood to include the value of any integerwithin the recited range and, when appropriate, fractions thereof (suchas one tenth and one hundredth of an integer), unless otherwiseindicated. Also, any number range recited herein relating to anyphysical feature, such as polymer subunits, size or thickness, are to beunderstood to include any integer within the recited range, unlessotherwise indicated. As used herein, “about” or “consisting essentiallyof” mean±20% of the indicated range, value, or structure, unlessotherwise indicated. As used herein, the terms “include” and “comprise”are open ended and are used synonymously.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present disclosure,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

As used herein, the term “nucleotide” refers to a ribonucleotide or adeoxyribonucleotide, or a modified form thereof, as well as an analogthereof. Nucleotides include species that include purines (e.g.,adenine, hypoxanthine, guanine, and their derivatives and analogs) aswell as pyrimidines (e.g., cytosine, uracil, thymine, and theirderivatives and analogs). As used herein, the term “cytidine” is usedgenerically to refer to a ribonucleotide, deoxyribonucleotide, ormodified ribonucleotide comprising a cytosine base, unless specificallyindicated otherwise. The term “cytidine” includes 2′-modified cytidines,such as 2′-fluoro, 2′-methoxy, etc. Similarly, the term “modifiedcytidine” or a specific modified cytidine also refers to aribonucleotide, deoxyribonucleotide, or modified ribonucleotide (such as2′-fluoro, 2′-methoxy, etc.) comprising the modified cytosine base,unless specifically indicated otherwise. The term “uridine” is usedgenerically to refer to a ribonucleotide, deoxyribonucleotide, ormodified ribonucleotide comprising a uracil base, unless specificallyindicated otherwise. The term “uridine” includes 2′-modified uridines,such as 2′-fluoro, 2′-methoxy, etc. Similarly, the term “modifieduridine” or a specific modified uridine also refers to a ribonucleotide,deoxyribonucleotide, or modified ribonucleotide (such as 2′-fluoro,2′-methoxy, etc.) comprising the modified uracil base, unlessspecifically indicated otherwise.

As used herein, the term “5-position modified cytidine” or “C-5 modifiedcytidine” refers to a cytidine with a modification at the C-5 positionof the cytidine, e.g., as shown in FIG. 20 . Nonlimiting exemplary5-position modified cytidines include those shown in FIG. 22 .Nonlimiting exemplary 5-position modified cytidines include, but are notlimited to, 5-(N-benzylcarboxamide)-2′-deoxycytidine (referred to as“BndC” and shown in FIG. 21 );5-(N-2-phenylethylcarboxamide)-2′-deoxycytidine (referred to as “PEdC”and shown in FIG. 21 ); 5-(N-3-phenylpropylcarboxamide)-2′-deoxycytidine(referred to as “PPdC” and shown in FIG. 21 );5-(N-1-naphthylmethylcarboxamide)-2′-deoxycytidine (referred to as“NapdC” and shown in FIG. 21 );5-(N-2-naphthylmethylcarboxamide)-2′-deoxycytidine (referred to as“2NapdC” and shown in FIG. 21 );5-(N-1-naphthyl-2-ethylcarboxamide)-2′-deoxycytidine (referred to as“NEdC” and shown in FIG. 21 );5-(N-2-naphthyl-2-ethylcarboxamide)-2′-deoxycytidine (referred to as“2NEdC” and shown in FIG. 21 ); and5-(N-tyrosylcarboxamide)-2′-deoxycytidine (referred to as TyrdC andshown in FIG. 21 ). In some embodiments, the C5-modified cytidines,e.g., in their triphosphate form, are capable of being incorporated intoan oligonucleotide by a polymerase (e.g., KOD DNA polymerase).

Chemical modifications of the C-5 modified cytidines described hereincan also be combined with, singly or in any combination, 2′-positionsugar modifications (for example, 2′-O-methyl or 2′-fluoro),modifications at exocyclic amines, and substitution of 4-thiocytidineand the like.

As used herein, the term “C-5 modified uridine” or “5-position modifieduridine” refers to a uridine (typically a deoxyuridine) with amodification at the C-5 position of the uridine, e.g., as shown in FIG.20 . In some embodiments, the C5-modified uridines, e.g., in theirtriphosphate form, are capable of being incorporated into anoligonucleotide by a polymerase (e.g., KOD DNA polymerase). Nonlimitingexemplary 5-position modified uridines include those shown in FIG. 21 .Nonlimiting exemplary 5-position modified uridines include:

-   5-(N-benzylcarboxamide)-2′-deoxyuridine (BndU),-   5-(N-phenethylcarboxamide)-2′-deoxyuridine (PEdU),-   5-(N-thiophenylmethylcarboxamide)-2′-deoxyuridine (ThdU),-   5-(N-isobutylcarboxamide)-2′-deoxyuridine (iBudU),-   5-(N-tyrosylcarboxamide)-2′-deoxyuridine (TyrdU),-   5-(N-3,4-methylenedioxybenzylcarboxamide)-2′-deoxyuridine (MBndU),-   5-(N-4-fluorobenzylcarboxamide)-2′-deoxyuridine (FBndU),-   5-(N-3-phenylpropylcarboxamide)-2′-deoxyuridine (PPdU),-   5-(N-imidizolylethylcarboxamide)-2′-deoxyuridine (ImdU),-   5-(N-tryptaminocarboxamide)-2′-deoxyuridine (TrpdU),-   5-(N—R-threoninylcarboxamide)-2′-deoxyuridine (ThrdU),-   5-(N-[1-(3-trimethylamonium) propyl]carboxamide)-2′-deoxyuridine    chloride,-   5-(N-naphthylmethylcarboxamide)-2′-deoxyuridine (NapdU),-   5-(N-[1-(2,3-dihydroxypropyl)]carboxamide)-2′-deoxyuridine),-   5-(N-2-naphthylmethylcarboxamide)-2′-deoxyuridine (2NapdU),-   5-(N-1-naphthylethylcarboxamide)-2′-deoxyuridine (NEdU),-   5-(N-2-naphthylethylcarboxamide)-2′-deoxyuridine (2NEdU),-   5-(N-3-benzofuranylethylcarboxamide)-2′-deoxyuridine (BFdU),-   5-(N-3-benzothiophenylethylcarboxamide)-2′-deoxyuridine (BTdU).

Chemical modifications of the C-5 modified uridines described herein canalso be combined with, singly or in any combination, 2′-position sugarmodifications (for example, 2′-O-methyl or 2′-fluoro), modifications atexocyclic amines, and substitution of 4-thiouridine and the like.

As used herein, the terms “modify,” “modified,” “modification,” and anyvariations thereof, when used in reference to an oligonucleotide, meansthat at least one of the four constituent nucleotide bases (i.e., A, G,T/U, and C) of the oligonucleotide is an analog or ester of a naturallyoccurring nucleotide. In some embodiments, the modified nucleotideconfers nuclease resistance to the oligonucleotide. Additionalmodifications can include backbone modifications, methylations, unusualbase-pairing combinations such as the isobases isocytidine andisoguanidine, and the like. Modifications can also include 3′ and 5′modifications, such as capping. Other modifications can includesubstitution of one or more of the naturally occurring nucleotides withan analog, internucleotide modifications such as, for example, thosewith uncharged linkages (e.g., methyl phosphonates, phosphotriesters,phosphoamidates, carbamates, etc.) and those with charged linkages(e.g., phosphorothioates, phosphorodithioates, etc.), those withintercalators (e.g., acridine, psoralen, etc.), those containingchelators (e.g., metals, radioactive metals, boron, oxidative metals,etc.), those containing alkylators, and those with modified linkages(e.g., alpha anomeric nucleic acids, etc.). Further, any of the hydroxylgroups ordinarily present on the sugar of a nucleotide may be replacedby a phosphonate group or a phosphate group; protected by standardprotecting groups; or activated to prepare additional linkages toadditional nucleotides or to a solid support. The 5′ and 3′ terminal OHgroups can be phosphorylated or substituted with amines, organic cappinggroup moieties of from about 1 to about 20 carbon atoms, polyethyleneglycol (PEG) polymers in one embodiment ranging from about 10 to about80 kDa, PEG polymers in another embodiment ranging from about 20 toabout 60 kDa, or other hydrophilic or hydrophobic biological orsynthetic polymers.

As used herein, “nucleic acid,” “oligonucleotide,” and “polynucleotide”are used interchangeably to refer to a polymer of nucleotides andinclude DNA, RNA, DNA/RNA hybrids and modifications of these kinds ofnucleic acids, oligonucleotides and polynucleotides, wherein theattachment of various entities or moieties to the nucleotide units atany position are included. The terms “polynucleotide,”“oligonucleotide,” and “nucleic acid” include double- or single-strandedmolecules as well as triple-helical molecules. Nucleic acid,oligonucleotide, and polynucleotide are broader terms than the termaptamer and, thus, the terms nucleic acid, oligonucleotide, andpolynucleotide include polymers of nucleotides that are aptamers but theterms nucleic acid, oligonucleotide, and polynucleotide are not limitedto aptamers.

Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including2′-O-methyl, 2′-O-allyl, 2′-O-ethyl, 2′-O-propyl, 2′-O—CH₂CH₂OCH₃,2′-fluoro, 2′-NH₂ or 2′-azido, carbocyclic sugar analogs, α-anomericsugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranosesugars, furanose sugars, sedoheptuloses, acyclic analogs and abasicnucleoside analogs such as methyl riboside. As noted herein, one or morephosphodiester linkages may be replaced by alternative linking groups.These alternative linking groups include embodiments wherein phosphateis replaced by P(O)S (“thioate”), P(S)S (“dithioate”), (O)NR^(X) ₂(“amidate”), P(O)R^(X), P(O)OR^(X)′, CO or CH₂ (“formacetal”), in whicheach R^(X) or R^(X)′ are independently H or substituted or unsubstitutedalkyl (C1-C20) optionally containing an ether (—O—) linkage, aryl,alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in apolynucleotide need be identical. Substitution of analogous forms ofsugars, purines, and pyrimidines can be advantageous in designing afinal product, as can alternative backbone structures like a polyamidebackbone, for example.

Polynucleotides can also contain analogous forms of carbocyclic sugaranalogs, α-anomeric sugars, epimeric sugars such as arabinose, xylosesor lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclicanalogs and abasic nucleoside analogs such as methyl riboside.

If present, a modification to the nucleotide structure can be impartedbefore or after assembly of a polymer. A sequence of nucleotides can beinterrupted by non-nucleotide components. A polynucleotide can befurther modified after polymerization, such as by conjugation with alabeling component.

As used herein, the term “at least one nucleotide” when referring tomodifications of a nucleic acid, refers to one, several, or allnucleotides in the nucleic acid, indicating that any or all occurrencesof any or all of A, C, T, G or U in a nucleic acid may be modified ornot.

As used herein, “nucleic acid ligand,” “aptamer,” “SOMAmer,” and “clone”are used interchangeably to refer to a non-naturally occurring nucleicacid that has a desirable action on a target molecule. A desirableaction includes, but is not limited to, binding of the target,catalytically changing the target, reacting with the target in a waythat modifies or alters the target or the functional activity of thetarget, covalently attaching to the target (as in a suicide inhibitor),and facilitating the reaction between the target and another molecule.In one embodiment, the action is specific binding affinity for a targetmolecule, such target molecule being a three dimensional chemicalstructure other than a polynucleotide that binds to the aptamer througha mechanism which is independent of Watson/Crick base pairing or triplehelix formation, wherein the aptamer is not a nucleic acid having theknown physiological function of being bound by the target molecule.Aptamers to a given target include nucleic acids that are identifiedfrom a candidate mixture of nucleic acids, where the aptamer is a ligandof the target, by a method comprising: (a) contacting the candidatemixture with the target, wherein nucleic acids having an increasedaffinity to the target relative to other nucleic acids in the candidatemixture can be partitioned from the remainder of the candidate mixture;(b) partitioning the increased affinity nucleic acids from the remainderof the candidate mixture; and (c) amplifying the increased affinitynucleic acids to yield a ligand-enriched mixture of nucleic acids,whereby aptamers of the target molecule are identified. It is recognizedthat affinity interactions are a matter of degree; however, in thiscontext, the “specific binding affinity” of an aptamer for its targetmeans that the aptamer binds to its target generally with a much higherdegree of affinity than it binds to other, non-target, components in amixture or sample. An “aptamer,” “SOMAmer,” or “nucleic acid ligand” isa set of copies of one type or species of nucleic acid molecule that hasa particular nucleotide sequence. An aptamer can include any suitablenumber of nucleotides. “Aptamers” refer to more than one such set ofmolecules. Different aptamers can have either the same or differentnumbers of nucleotides. Aptamers may be DNA or RNA and may be singlestranded, double stranded, or contain double stranded or triple strandedregions. In some embodiments, the aptamers are prepared using a SELEXprocess as described herein, or known in the art.

As used herein, a “SOMAmer” or Slow Off-Rate Modified Aptamer refers toan aptamer having improved off-rate characteristics. SOMAmers can begenerated using the improved SELEX methods described in U.S. Pat. No.7,947,447, entitled “Method for Generating Aptamers with ImprovedOff-Rates.”

As used herein, an aptamer comprising two different types of 5-positionmodified pyrimidines or C-5 modified pyrimidines may be referred to as“dual modified aptamers”, aptamers having “two modified bases”, aptamershaving “two base modifications” or “two bases modified”, aptamer having“double modified bases”, all of which may be used interchangeably. Alibrary of aptamers or aptamer library may also use the sameterminology. Thus, in some embodiments, an aptamer comprises twodifferent 5-position modified pyrimidines wherein the two different5-position modified pyrimidines are selected from a NapdC and a NapdU, aNapdC and a PPdU, a NapdC and a MOEdU, a NapdC and a TyrdU, a NapdC anda ThrdU, a PPdC and a PPdU, a PPdC and a NapdU, a PPdC and a MOEdU, aPPdC and a TyrdU, a PPdC and a ThrdU, a NapdC and a 2NapdU, a NapdC anda TrpdU, a 2NapdC and a NapdU, and 2NapdC and a 2NapdU, a 2NapdC and aPPdU, a 2NapdC and a TrpdU, a 2NapdC and a TyrdU, a PPdC and a 2NapdU, aPPdC and a TrpdU, a PPdC and a TyrdU, a TyrdC and a TyrdU, a TrydC and a2NapdU, a TyrdC and a PPdU, a TyrdC and a TrpdU, a TyrdC and a TyrdU,and a TyrdC and a TyrdU. In some embodiments, an aptamer comprises atleast one modified uridine and/or thymidine and at least one modifiedcytidine, wherein the at least one modified uridine and/or thymidine ismodified at the 5-position with a moiety selected from a naphthylmoiety, a benzyl moiety, a fluorobenzyl moiety, a tyrosyl moiety, anindole moiety a morpholino moiety, an isobutyl moiety, a3,4-methylenedioxy benzyl moiety, a benzothiophenyl moiety, and abenzofuranyl moiety, and wherein the at least one modified cytidine ismodified at the 5-position with a moiety selected from a naphthylmoiety, a tyrosyl moiety, and a benzyl moiety. In certain embodiments,the moiety is covalently linked to the 5-position of the base via alinker comprising a group selected from an amide linker, a carbonyllinker, a propynyl linker, an alkyne linker, an ester linker, a urealinker, a carbamate linker, a guanidine linker, an amidine linker, asulfoxide linker, and a sulfone linker.

As used herein, an aptamer comprising a single type of 5-positionmodified pyrimidine or C-5 modified pyrimidine may be referred to as“single modified aptamers”, aptamers having a “single modified base”,aptamers having a “single base modification” or “single bases modified”,all of which may be used interchangeably. A library of aptamers oraptamer library may also use the same terminology. As used herein,“protein” is used synonymously with “peptide,” “polypeptide,” or“peptide fragment.” A “purified” polypeptide, protein, peptide, orpeptide fragment is substantially free of cellular material or othercontaminating proteins from the cell, tissue, or cell-free source fromwhich the amino acid sequence is obtained, or substantially free fromchemical precursors or other chemicals when chemically synthesized.

In certain embodiments, an aptamer comprises a first 5-position modifiedpyrimidine and a second 5-position modified pyrimidine, wherein thefirst 5-position modified pyrimidine comprises a tyrosyl moiety at the5-position of the first 5-position modified pyrimidine, and the second5-position modified pyrimidine comprises a naphthyl moiety or benzylmoiety at the 5-position at the second 5-position modified pyrimidine.In a related embodiment the first 5-position modified pyrimidine is auracil. In a related embodiment, the second 5-position modifiedpyrimidine is a cytosine. In a related embodiment, at least 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% of the uracils of the aptamer are modified at the5-position. In a related embodiment, at least 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%of the cytosine of the aptamer are modified at the 5-position.

Modified Nucleotides

In certain embodiments, the disclosure provides oligonucleotides, suchas aptamers, which comprise two different types of base-modifiednucleotides. In some embodiments, the oligonucleotides comprise twodifferent types of 5-position modified pyrimidines. In some embodiments,the oligonucleotide comprises at least one C5-modified cytidine and atleast one C5-modified uridine. In some embodiments, the oligonucleotidecomprises two different C5-modified cytidines. In some embodiments, theoligonucleotide comprises two different C5-modified uridines.Nonlimiting exemplary C5-modified uridines and cytidines are shown, forexample, in Formula I below, and in FIG. 20 . Certain nonlimitingexemplary C5-modified uridines are shown in FIG. 21 , and certainnonlimiting exemplary C5-modified cytidines are shown in FIG. 22 .

In some embodiments, the oligonucleotide comprises at least onepyrimidine of Formula I:

wherein

-   -   R is independently a —(CH₂)_(n)—, wherein n is an integer        selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;    -   R^(X1) is independently selected from the group consisting of

wherein,

denotes the point of attachment of the R^(X1) group to the —(CH₂)_(n)—group; and wherein,

-   -   R^(X4) is independently selected from the group consisting of a        branched or linear lower alkyl (C1-C20); a hydroxyl group; a        halogen (F, Cl, Br, I); nitrile (CN); boronic acid (BO₂H₂);        carboxylic acid (COOH); carboxylic acid ester (COOR^(X2));        primary amide (CONH₂); secondary amide (CONHR^(X2)); tertiary        amide (CONR^(X2)R^(X3)); sulfonamide (SO₂NH₂);        N-alkylsulfonamide (SONHR^(X2));    -   R^(X2) and R^(X3) are independently, for each occurrence,        selected from the group consisting of a branched or linear lower        alkyl (C1-C20); phenyl (C₆H₅); an R^(X4) substituted phenyl ring        (R^(X4)C₆H₄), wherein R^(X4) is defined above; a carboxylic acid        (COOH); a carboxylic acid ester (COOR^(X5)), wherein R^(X5) is a        branched or linear lower alkyl (C1-C20); and cycloalkyl, wherein        R^(X2) and R^(X3) together form a substituted or unsubstituted 5        or 6 membered ring;    -   X is independently selected from the group consisting of —H,        —OH, —OMe, —O-allyl, —F, —OEt, —OPr, —OCH₂CH₂OCH₃, NH₂ and        -azido;    -   R′ is independently selected from the group consisting of a —H,        —OAc; —OBz; —P(NiPr₂)(OCH₂CH₂CN); and —OSiMe₂tBu;    -   R″ is independently selected from the group consisting of a        hydrogen, 4,4′-dimethoxytrityl (DMT) and triphosphate        (—P(O)(OH)—O—P(O)(OH)—O—P(O)(OH)₂) or a salt thereof;    -   Z is independently selected from the group consisting of a —H, a        substituted or unsubstituted C(1-4)alkyl;        and salts thereof.

In some embodiments, the oligonucleotide comprises at least one modifiedpyrimidine shown in FIG. 21 , wherein each X is independently selectedfrom —H, —OH, —OMe, —O-allyl, —F, —OEt, —OPr, —OCH₂CH₂OCH₃, NH₂ and-azido.

In some embodiments, the oligonucleotide comprises at least one modifiedpyrimidine shown in FIG. 22 , wherein each X is independently selectedfrom —H, —OH, —OMe, —O-allyl, —F, —OEt, —OPr, —OCH₂CH₂OCH₃, NH₂ and-azido.

In some embodiments, the oligonucleotide comprises at least one modifiedpyrimidine shown in FIG. 21 and at least one modified pyrimidine shownin FIG. 22 , wherein each X is independently selected from —H, —OH,—OMe, —O-allyl, —F, —OEt, —OPr, —OCH₂CH₂OCH₃, NH₂ and -azido. Certainnonlimiting exemplary pairs of modified pyrimidines are shown in theExamples described herein.

In some embodiments, the oligonucleotide comprises at least one modifiedpyrimidine shown in FIG. 20 , wherein the 2′ position of the ribose isindependently selected from —H, —OH, —OMe, —O-allyl, —F, —OEt, —OPr,—OCH₂CH₂OCH₃, NH₂ and -azido. In some embodiments, the oligonucleotidecomprises at least two modified pyrimidines shown in FIG. 20 , whereinthe 2′ position of the ribose is independently selected from —H, —OH,—OMe, —O-allyl, —F, —OEt, —OPr, —OCH₂CH₂OCH₃, NH₂ and -azido.

In some embodiments, the oligonucleotide comprises at least one modifiedpyrimidine shown in FIG. 20 and at least one modified pyrimidine shownin FIG. 21 or FIG. 22 , wherein the 2′ position of the ribose isindependently selected from —H, —OH, —OMe, —O-allyl, —F, —OEt, —OPr,—OCH₂CH₂OCH₃, NH₂ and -azido. Certain nonlimiting exemplary pairs ofmodified pyrimidines are shown in the Examples described herein.

In any of the embodiments described herein, the oligonucleotide may bean aptamer. In some such embodiments, the oligonucleotide is an aptamerthat specifically binds a target polypeptide.

Preparation of Oligonucleotides

The automated synthesis of oligodeoxynucleosides is routine practice inmany laboratories (see e.g., Matteucci, M. D. and Caruthers, M. H.,(1990) J. Am. Chem. Soc., 103:3185-3191, the contents of which arehereby incorporated by reference in their entirety). Synthesis ofoligoribonucleosides is also well known (see e.g. Scaringe, S. A., etal., (1990) Nucleic Acids Res. 18:5433-5441, the contents of which arehereby incorporated by reference in their entirety). As noted herein,the phosphoramidites are useful for incorporation of the modifiednucleoside into an oligonucleotide by chemical synthesis, and thetriphosphates are useful for incorporation of the modified nucleosideinto an oligonucleotide by enzymatic synthesis. (See e.g., Vaught, J. D.et al. (2004) J. Am. Chem. Soc., 126:11231-11237; Vaught, J. V., et al.(2010) J. Am. Chem. Soc. 132, 4141-4151; Gait, M. J. “OligonucleotideSynthesis a practical approach” (1984) IRL Press (Oxford, UK);Herdewijn, P. “Oligonucleotide Synthesis” (2005) (Humana Press, Totowa,N.J. (each of which is incorporated herein by reference in itsentirety).

The SELEX Method

The terms “SELEX” and “SELEX process” are used interchangeably herein torefer generally to a combination of (1) the selection of nucleic acidsthat interact with a target molecule in a desirable manner, for examplebinding with high affinity to a protein, with (2) the amplification ofthose selected nucleic acids. The SELEX process can be used to identifyaptamers with high affinity to a specific target molecule or biomarker.

SELEX generally includes preparing a candidate mixture of nucleic acids,binding of the candidate mixture to the desired target molecule to forman affinity complex, separating the affinity complexes from the unboundcandidate nucleic acids, separating and isolating the nucleic acid fromthe affinity complex, purifying the nucleic acid, and identifying aspecific aptamer sequence. The process may include multiple rounds tofurther refine the affinity of the selected aptamer. The process caninclude amplification steps at one or more points in the process. See,e.g., U.S. Pat. No. 5,475,096, entitled “Nucleic Acid Ligands.” TheSELEX process can be used to generate an aptamer that covalently bindsits target as well as an aptamer that non-covalently binds its target.See, e.g., U.S. Pat. No. 5,705,337 entitled “Systematic Evolution ofNucleic Acid Ligands by Exponential Enrichment: Chemi-SELEX.”

The SELEX process can be used to identify high-affinity aptamerscontaining modified nucleotides that confer improved characteristics onthe aptamer, such as, for example, improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX process-identified aptamers containing modifiednucleotides are described in U.S. Pat. No. 5,660,985, entitled “HighAffinity Nucleic Acid Ligands Containing Modified Nucleotides,” whichdescribes oligonucleotides containing nucleotide derivatives chemicallymodified at the 5′- and 2′-positions of pyrimidines. U.S. Pat. No.5,580,737, see supra, describes highly specific aptamers containing oneor more nucleotides modified with 2′-amino (2′-NH₂), 2′-fluoro (2′-F),and/or 2′-O-methyl (2′-OMe). See also, U.S. Patent ApplicationPublication No. 20090098549, entitled “SELEX and PHOTOSELEX,” whichdescribes nucleic acid libraries having expanded physical and chemicalproperties and their use in SELEX and photoSELEX.

SELEX can also be used to identify aptamers that have desirable off-ratecharacteristics. See U.S. Pat. No. 7,947,447, entitled “Method forGenerating Aptamers with Improved Off-Rates,” which is incorporatedherein by reference in its entirety, describes improved SELEX methodsfor generating aptamers that can bind to target molecules. Methods forproducing aptamers and photoaptamers having slower rates of dissociationfrom their respective target molecules are described. The methodsinvolve contacting the candidate mixture with the target molecule,allowing the formation of nucleic acid-target complexes to occur, andperforming a slow off-rate enrichment process wherein nucleicacid-target complexes with fast dissociation rates dissociate and do notreform, while complexes with slow dissociation rates remain intact.Additionally, the methods include the use of modified nucleotides in theproduction of candidate nucleic acid mixtures to generate aptamers withimproved off-rate performance (see U.S. Pat. No. 8,409,795, entitled“SELEX and PhotoSELEX”). (See also U.S. Pat. No. 7,855,054 and U.S.Patent Publication No. 20070166740). Each of these applications isincorporated herein by reference in its entirety.

“Target” or “target molecule” or “target” refers herein to any compoundupon which a nucleic acid can act in a desirable manner. A targetmolecule can be a protein, peptide, nucleic acid, carbohydrate, lipid,polysaccharide, glycoprotein, hormone, receptor, antigen, antibody,virus, pathogen, toxic substance, substrate, metabolite, transitionstate analog, cofactor, inhibitor, drug, dye, nutrient, growth factor,cell, tissue, any portion or fragment of any of the foregoing, etc.,without limitation. Virtually any chemical or biological effector may bea suitable target. Molecules of any size can serve as targets. A targetcan also be modified in certain ways to enhance the likelihood orstrength of an interaction between the target and the nucleic acid. Atarget can also include any minor variation of a particular compound ormolecule, such as, in the case of a protein, for example, minorvariations in amino acid sequence, disulfide bond formation,glycosylation, lipidation, acetylation, phosphorylation, or any othermanipulation or modification, such as conjugation with a labelingcomponent, which does not substantially alter the identity of themolecule. A “target molecule” or “target” is a set of copies of one typeor species of molecule or multimolecular structure that is capable ofbinding to an aptamer. “Target molecules” or “targets” refer to morethan one such set of molecules. Embodiments of the SELEX process inwhich the target is a peptide are described in U.S. Pat. No. 6,376,190,entitled “Modified SELEX Processes Without Purified Protein.” In someembodiments, a target is a protein.

As used herein, “competitor molecule” and “competitor” are usedinterchangeably to refer to any molecule that can form a non-specificcomplex with a non-target molecule. In this context, non-targetmolecules include free aptamers, where, for example, a competitor can beused to inhibit the aptamer from binding (rebinding), non-specifically,to another non-target molecule. A “competitor molecule” or “competitor”is a set of copies of one type or species of molecule. “Competitormolecules” or “competitors” refer to more than one such set ofmolecules. Competitor molecules include, but are not limited tooligonucleotides, polyanions (e.g., heparin, herring sperm DNA, salmonsperm DNA, tRNA, dextran sulfate, polydextran, abasic phosphodiesterpolymers, dNTPs, and pyrophosphate). In various embodiments, acombination of one or more competitor can be used.

As used herein, “non-specific complex” refers to a non-covalentassociation between two or more molecules other than an aptamer and itstarget molecule. A non-specific complex represents an interactionbetween classes of molecules. Non-specific complexes include complexesformed between an aptamer and a non-target molecule, a competitor and anon-target molecule, a competitor and a target molecule, and a targetmolecule and a non-target molecule.

As used herein, the term “slow off-rate enrichment process” refers to aprocess of altering the relative concentrations of certain components ofa candidate mixture such that the relative concentration of aptameraffinity complexes having slow dissociation rates is increased relativeto the concentration of aptamer affinity complexes having faster, lessdesirable dissociation rates. In one embodiment, the slow off-rateenrichment process is a solution-based slow off-rate enrichment process.In this embodiment, a solution-based slow off-rate enrichment processtakes place in solution, such that neither the target nor the nucleicacids forming the aptamer affinity complexes in the mixture areimmobilized on a solid support during the slow off-rate enrichmentprocess. In various embodiments, the slow-off rate enrichment processcan include one or more steps, including the addition of and incubationwith a competitor molecule, dilution of the mixture, or a combination ofthese (e.g., dilution of the mixture in the presence of a competitormolecule). Because the effect of an slow off-rate enrichment processgenerally depends upon the differing dissociation rates of differentaptamer affinity complexes (i.e., aptamer affinity complexes formedbetween the target molecule and different nucleic acids in the candidatemixture), the duration of the slow off-rate enrichment process isselected so as to retain a high proportion of aptamer affinity complexeshaving slow dissociation rates while substantially reducing the numberof aptamer affinity complexes having fast dissociation rates. The slowoff-rate enrichment process may be used in one or more cycles during theSELEX process. When dilution and the addition of a competitor are usedin combination, they may be performed simultaneously or sequentially, inany order. The slow-off rate enrichment process can be used when thetotal target (protein) concentration in the mixture is low. In oneembodiment, when the slow off-rate enrichment process includes dilution,the mixture can be diluted as much as is practical, keeping in mind thatthe aptamer retained nucleic acids are recovered for subsequent roundsin the SELEX process. In one embodiment, the slow off-rate enrichmentprocess includes the use of a competitor as well as dilution, permittingthe mixture to be diluted less than might be necessary without the useof a competitor.

In one embodiment, the slow off-rate enrichment process includes theaddition of a competitor, and the competitor is a polyanion (e.g.,heparin or dextran sulfate (dextran)). Heparin or dextran have been usedin the identification of specific aptamers in prior SELEX selections. Insuch methods, however, heparin or dextran is present during theequilibration step in which the target and aptamer bind to formcomplexes. In such methods, as the concentration of heparin or dextranincreases, the ratio of high affinity target/aptamer complexes to lowaffinity target/aptamer complexes increases. However, a highconcentration of heparin or dextran can reduce the number of highaffinity target/aptamer complexes at equilibrium due to competition fortarget binding between the nucleic acid and the competitor. By contrast,the presently described methods add the competitor after thetarget/aptamer complexes have been allowed to form and therefor does notaffect the number of complexes formed. Addition of competitor afterequilibrium binding has occurred between target and aptamer creates anon-equilibrium state that evolves in time to a new equilibrium withfewer target/aptamer complexes. Trapping target/aptamer complexes beforethe new equilibrium has been reached enriches the sample for slowoff-rate aptamers since fast off-rate complexes will dissociate first.

In another embodiment, a polyanionic competitor (e.g., dextran sulfateor another polyanionic material) is used in the slow off-rate enrichmentprocess to facilitate the identification of an aptamer that isrefractory to the presence of the polyanion. In this context,“polyanionic refractory aptamer” is an aptamer that is capable offorming an aptamer/target complex that is less likely to dissociate inthe solution that also contains the polyanionic refractory material thanan aptamer/target complex that includes a nonpolyanionic refractoryaptamer. In this manner, polyanionic refractory aptamers can be used inthe performance of analytical methods to detect the presence or amountor concentration of a target in a sample, where the detection methodincludes the use of the polyanionic material (e.g. dextran sulfate) towhich the aptamer is refractory.

Thus, in one embodiment, a method for producing a polyanionic refractoryaptamer is provided. In this embodiment, after contacting a candidatemixture of nucleic acids with the target. The target and the nucleicacids in the candidate mixture are allowed to come to equilibrium. Apolyanionic competitor is introduced and allowed to incubate in thesolution for a period of time sufficient to insure that most of the fastoff rate aptamers in the candidate mixture dissociate from the targetmolecule. Also, aptamers in the candidate mixture that may dissociate inthe presence of the polyanionic competitor will be released from thetarget molecule. The mixture is partitioned to isolate the highaffinity, slow off-rate aptamers that have remained in association withthe target molecule and to remove any uncomplexed materials from thesolution. The aptamer can then be released from the target molecule andisolated. The isolated aptamer can also be amplified and additionalrounds of selection applied to increase the overall performance of theselected aptamers. This process may also be used with a minimalincubation time if the selection of slow off-rate aptamers is not neededfor a specific application.

Thus, in one embodiment a modified SELEX process is provided for theidentification or production of aptamers having slow (long) off rateswherein the target molecule and candidate mixture are contacted andincubated together for a period of time sufficient for equilibriumbinding between the target molecule and nucleic acids contained in thecandidate mixture to occur. Following equilibrium binding an excess ofcompetitor molecule, e.g., polyanion competitor, is added to the mixtureand the mixture is incubated together with the excess of competitormolecule for a predetermined period of time. A significant proportion ofaptamers having off rates that are less than this predeterminedincubation period will dissociate from the target during thepredetermined incubation period. Re-association of these “fast” off rateaptamers with the target is minimized because of the excess ofcompetitor molecule which can non-specifically bind to the target andoccupy target binding sites. A significant proportion of aptamers havinglonger off rates will remain complexed to the target during thepredetermined incubation period. At the end of the incubation period,partitioning nucleic acid-target complexes from the remainder of themixture allows for the separation of a population of slow off-rateaptamers from those having fast off rates. A dissociation step can beused to dissociate the slow off-rate aptamers from their target andallows for isolation, identification, sequencing, synthesis andamplification of slow off-rate aptamers (either of individual aptamersor of a group of slow off-rate aptamers) that have high affinity andspecificity for the target molecule. As with conventional SELEX theaptamer sequences identified from one round of the modified SELEXprocess can be used in the synthesis of a new candidate mixture suchthat the steps of contacting, equilibrium binding, addition ofcompetitor molecule, incubation with competitor molecule andpartitioning of slow off-rate aptamers can be iterated/repeated as manytimes as desired.

The combination of allowing equilibrium binding of the candidate mixturewith the target prior to addition of competitor, followed by theaddition of an excess of competitor and incubation with the competitorfor a predetermined period of time allows for the selection of apopulation of aptamers having off rates that are much greater than thosepreviously achieved.

In order to achieve equilibrium binding, the candidate mixture may beincubated with the target for at least about 5 minutes, or at leastabout 15 minutes, about 30 minutes, about 45 minutes, about 1 hour,about 2 hours, about 3 hours, about 4 hours, about 5 hours or about 6hours.

The predetermined incubation period of competitor molecule with themixture of the candidate mixture and target molecule may be selected asdesired, taking account of the factors such as the nature of the targetand known off rates (if any) of known aptamers for the target.Predetermined incubation periods may be chosen from: at least about 5minutes, at least about 10 minutes, at least about 20 minutes, at leastabout 30 minutes, at least 45 about minutes, at least about 1 hour, atleast about 2 hours, at least about 3 hours, at least about 4 hours, atleast about 5 hours, at least about 6 hours.

In other embodiments a dilution is used as an off rate enhancementprocess and incubation of the diluted candidate mixture, targetmolecule/aptamer complex may be undertaken for a predetermined period oftime, which may be chosen from: at least about 5 minutes, at least about10 minutes, at least about 20 minutes, at least about 30 minutes, atleast about 45 minutes, at least about 1 hour, at least about 2 hours,at least about 3 hours, at least about 4 hours, at least about 5 hours,at least about 6 hours.

Embodiments of the present disclosure are concerned with theidentification, production, synthesis and use of slow off-rate aptamers.These are aptamers which have a rate of dissociation (t_(1/2)) from anon-covalent aptamer-target complex that is higher than that of aptamersnormally obtained by conventional SELEX. For a mixture containingnon-covalent complexes of aptamer and target, the t_(1/2) represents thetime taken for half of the aptamers to dissociate from theaptamer-target complexes. The t_(1/2) of slow dissociation rate aptamersaccording to the present disclosure is chosen from one of: greater thanor equal to about 30 minutes; between about 30 minutes and about 240minutes; between about 30 minutes to about 60 minutes; between about 60minutes to about 90 minutes, between about 90 minutes to about 120minutes; between about 120 minutes to about 150 minutes; between about150 minutes to about 180 minutes; between about 180 minutes to about 210minutes; between about 210 minutes to about 240 minutes.

A characterizing feature of an aptamer identified by a SELEX procedureis its high affinity for its target. An aptamer will have a dissociationconstant (k_(d)) for its target that is chosen from one of: less thanabout 1 μM, less than about 100 nM, less than about 10 nM, less thanabout 1 nM, less than about 100 pM, less than about 10 pM, less thanabout 1 pM.

Libraries of Oligonucleotides

In some embodiments, libraries of oligonucleotides comprising randomsequences are provided. Such libraries may be useful, in someembodiments, for performing SELEX. In some embodiments, eacholigonucleotide of a library of oligonucleotides comprises a number ofrandomized positions, such as at least 20, 25, 30, 35, 40, 45, or 50, or20-100, 20-80, 20-70, 20-60, 20-50, 20-40, or 30-40 randomizedpositions. In some embodiments, each oligonucleotide of a library ofoligonucleotides comprises fixed sequences flanking the randomizedpositions. Such fixed flanking sequences may be the same or differentfrom one another (i.e., the 5′ flanking sequence and the 3′ flankingsequence may be the same or different), and may, in some embodiments, bethe same for all members of the library (i.e., all members of thelibrary may have the same 5′ flanking sequence, and/or all members ofthe library may have the same 3′ flanking sequence).

In some embodiments, the randomized positions may be made up of four ormore different nucleotide bases, one or more of which is modified. Insome embodiments, all of one type of nucleotide base is modified orunmodified (e.g., all of the cytidines in the randomized region ormodified, or all are unmodified). In some embodiments, one type ofnucleotide base in the randomized region is present in both modified andunmodified forms. In some such embodiments, the randomized positions aremade up of two modified and two unmodified nucleotide bases. In somesuch embodiments, the randomized positions are made up of adenine,guanine, C5-modified cytidine, and C5-modified uridine. Nonlimitingexemplary C5-modified cytidines and C5-modified uridines are shown inFIGS. 19 to 21 . Libraries of oligonucleotides and methods of makingthem are further described, e.g., in the Examples herein.

Exemplary Aptamers

In some embodiments, aptamers that bind a target molecule are provided.In some embodiments, the target molecule is a target protein. In someembodiments, aptamers that bind PCSK9 are provided. In some embodiments,an aptamer that binds PCSK9 inhibits binding of PCSK9 to LDL-R. In somesuch embodiments, the aptamer comprises the sequence 5′-yGpppG-3′,wherein each y is a TyrdU and each p is a NapdC. In some embodiments,the aptamer further comprises the sequence 5′-yEAyGA_(n)pAp-3′, whereinE is selected from y, A, and G; and n is 0 or 1. In some embodiments, nis 0. In some embodiments, the sequence 5′-yEAyGA_(n)pAp-3′ is located5′ of the sequence 5′-yGpppG-3′. In some embodiments, E is y.

In some embodiments, an aptamer that binds PCSK9 is provided, whereinthe aptamer comprises the sequence 5′-F_(n)pppAAGRJrpRppW_(m)-3′ (SEQ IDNO: 81), wherein F is selected from r and G; each R is independentlyselected from G and A; J is selected from r and A; W is selected from r,G, and A; n is 0 or 1; m is 0 or 1; r is PpdC; and p is NapdU. In someembodiments, m is 1. In some embodiments, F is r. In some embodiments, Jis r. In some embodiments, W is G.

In some embodiments, an aptamer that binds PCSK9 is provided, whereinthe aptamer comprises the sequence 5′-TTppGGpp-3′, wherein each p is aNapdC.

In some embodiments, an aptamer that binds PCSK9 is 20 to 100, or 20 to90, or 20 to 80, or 20 to 70, or 20 to 60, or 20 to 50, or 30 to 100, or30 to 90, or 30 to 80, or 30 to 70, or 30 to 60, or 30 to 50, or 40 to100, or 40 to 90, or 40 to 80, or 40 to 70, or 40 to 60, or 40 to 50nucleotides in length.

In some embodiments, the aptamer inhibits PCSK9 binding to LDL-R. Insome embodiments, the aptamer inhibits PCSK9 binding to LDL-R with anIC₅₀ of less than 30 nM, less than 20 nM, or less than 15 nM.

In some embodiments, a method of lowering cholesterol in a subject isprovided, comprising administering to a subject in need thereof anaptamer that binds PCSK9. In some embodiments, the aptamer that bindsPCSK9 is an aptamer provided herein. In some embodiments, thecholesterol is low-density lipoprotein (LDL) cholesterol (LDL-C). Insome embodiments, the subject has heterozygous familialhypercholesterolemia or clinical atherosclerotic cardiovascular disease(CVD).

Salts

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge et al. (1977) “PharmaceuticallyAcceptable Salts” J. Pharm. Sci. 66:1-19.

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g., —COOH may be —COO⁻), then a salt may be formedwith a suitable cation. Examples of suitable inorganic cations include,but are not limited to, alkali metal ions such as Na⁺ and K⁺, alkalineearth cations such as Ca²⁺ and Mg²⁺, and other cations such as Al⁺³.Examples of suitable organic cations include, but are not limited to,ammonium ion (i.e., NH₄ ⁺) and substituted ammonium ions (e.g.,NH₃R^(X+), NH₂R^(X) ₂ ⁺, NHR^(X) ₃ ⁺, NR^(X) ₄ ⁺). Examples of somesuitable substituted ammonium ions are those derived from: ethylamine,diethylamine, dicyclohexylamine, triethylamine, butylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

If the compound is cationic, or has a functional group which may becationic (e.g., —NH₂ may be —NH₃ ⁺), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to,those derived from the following organic acids: 2-acetyoxybenzoic,acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric,edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucoheptonic,gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic,methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic,phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic,succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examplesof suitable polymeric organic anions include, but are not limited to,those derived from the following polymeric acids: tannic acid,carboxymethyl cellulose.

Unless otherwise specified, a reference to a particular compound alsoincludes salt forms thereof.

Certain Nonlimiting Exemplary Embodiments

Embodiment 1. An aptamer comprising at least one first 5-positionmodified pyrimidine and at least one second 5-position modifiedpyrimidine, wherein the first 5-position modified pyrimidine and thesecond 5-position modified pyrimidine are different 5-position modifiedpyrimidines.

Embodiment 2. The aptamer of embodiment 1, wherein the first 5-positionmodified pyrimidine is a 5-position modified uridine and wherein thesecond 5-position modified pyrimidine is a 5-position modified cytidine.

Embodiment 3. The aptamer of embodiment 1, wherein the first 5-positionmodified pyrimidine is a 5-position modified cytidine and wherein thesecond 5-position modified pyrimidine is a 5-position modified uridine.

Embodiment 4. The aptamer of embodiment 2 or embodiment 3, wherein the5-position modified uridine comprises a moiety at the 5-positionselected from a naphthyl moiety, a benzyl moiety, a tyrosyl moiety, anindole moiety and a morpholino moiety.

Embodiment 5. The aptamer of any one of embodiments 2 to 4, wherein the5-position modified cytidine comprises a moiety at the 5-positionselected from a naphthyl moiety, a benzyl moiety, a tyrosyl moiety, anda morpholino moiety.

Embodiment 6. The aptamer of any one of embodiments 2 to 5, wherein the5-position modified cytidine is selected from a NapdC, a 2NapdC, aTyrdC, and a PPdC.

Embodiment 7. The aptamer of any one of embodiments 2 to 6, wherein the5-position modified uridine is selected from a NapdU, a 2NapdU, a PPdU,a MOEdU, a TyrdU, a TrpdU, and a ThrdU.

Embodiment 8. The aptamer of embodiment 1, wherein the at least onefirst 5-position modified pyrimidine is a NapdC and the at least onesecond 5-position modified pyrimidine is selected from a NapdU, a2NapdU, a PPdU, a MOEdU, a TyrdU, and a ThrdU.

Embodiment 9. The aptamer of embodiment 1, wherein the at least onefirst 5-position modified pyrimidine is a PPdC and the at least onesecond 5-position modified pyrimidine is selected from a NapdU, a2NapdU, a PPdU, a MOEdU, a TyrdU, and a ThrdU.

Embodiment 10. The aptamer of embodiment 8 or embodiment 9, wherein theat least one second 5-position modified pyrimidine is a TyrdU.

Embodiment 11. The aptamer of any one of embodiments 1 to 10, whereinthe aptamer binds a target protein selected from PCSK9, PSMA, ErbB1,ErbB2, FXN, KDM2A, IGF1R, pIGF1R, al-Antritrypsin, CD99, MMP28 and PPIB.

Embodiment 12. The aptamer of any one of embodiments 1 to 11, whereinthe aptamer comprises a region at the 5′ end of the aptamer that is atleast 10, at least 15, at least 20, at least 25 or at least 30nucleotides in length, or 5 to 30, 10 to 30, 15 to 30, 5 to 20, or 10 to20 nucleotides in length, wherein the region at the 5′ end of theaptamer lacks 5-position modified pyrimidines.

Embodiment 13. The aptamer of any one of embodiments 1 to 12, whereinthe aptamer comprises a region at the 3′ end of the aptamer that is atleast 10, at least 15, at least 20, at least 25 or at least 30nucleotides in length, or 5 to 30, 10 to 30, 15 to 30, 5 to 20, or 10 to20 nucleotides in length, wherein the region at the 3′ end of theaptamer lacks 5-position modified pyrimidines.

Embodiment 14. The aptamer of any one of embodiments 1 to 13, whereinthe aptamer is 20 to 100, or 20 to 90, or 20 to 80, or 20 to 70, or 20to 60, or 20 to 50, or 30 to 100, or 30 to 90, or 30 to 80, or 30 to 70,or 30 to 60, or 30 to 50, or 40 to 100, or 40 to 90, or 40 to 80, or 40to 70, or 40 to 60, or 40 to 50 nucleotides in length.

Embodiment 15. A composition comprising a plurality of polynucleotides,wherein each polynucleotide comprises at least one first 5-positionmodified pyrimidine and at least one second 5-position modifiedpyrimidine, wherein the first 5-position modified pyrimidine and thesecond 5-position modified pyrimidine are different 5-position modifiedpyrimidines.

Embodiment 16. The composition of embodiment 15, wherein eachpolynucleotide comprises a fixed region at the 5′ end of thepolynucleotide.

Embodiment 17. The composition of embodiment 16, wherein the fixedregion at the 5′ end of each polynucleotide is at least 10, at least 15,at least 20, at least 25 or at least 30 nucleotides in length, or 5 to30, 10 to 30, 15 to 30, 5 to 20, or 10 to 20 nucleotides in length.

Embodiment 18. The composition of any one of embodiments 15 to 17,wherein each polynucleotide comprises a fixed region at the 3′ end ofthe polynucleotide.

Embodiment 19. The composition of embodiment 18, wherein the fixedregion at the 3′ end of the polynucleotide is at least 10, at least 15,at least 20, at least 25 or at least 30 nucleotides in length, or 5 to30, 10 to 30, 15 to 30, 5 to 20, or 10 to 20 nucleotides in length.

Embodiment 20. The composition of any one of embodiments 15 to 19,wherein the first 5-position modified pyrimidine is a 5-positionmodified uridine and wherein the second 5-position modified pyrimidineis a 5-position modified cytidine.

Embodiment 21. The composition of any one of embodiments 15 to 19,wherein the first 5-position modified pyrimidine is a 5-positionmodified cytidine and wherein the second 5-position modified pyrimidineis a 5-position modified uridine.

Embodiment 22. The composition of embodiment 20 or embodiment 21,wherein the 5-position modified uridine comprises a moiety at the5-position selected from a naphthyl moiety, a benzyl moiety, a tyrosylmoiety, an indole moiety and a morpholino moiety.

Embodiment 23. The composition of any one of embodiments 20 to 22,wherein the 5-position modified cytidine comprises a moiety at the5-position selected from a naphthyl moiety, a benzyl moiety, a tyrosylmoiety, and a morpholino moiety.

Embodiment 24. The composition of any one of embodiments 20 to 23,wherein the 5-position modified cytidine is selected from NapdC, 2NapdC,TyrdC, and PPdC.

Embodiment 25. The composition of any one of embodiments 20 to 24,wherein the 5-position modified uridine is selected from NapdU, 2NapdU,PPdU, MOEdU, TyrdU, TrpdU, and ThrdU.

Embodiment 26. The composition of embodiment 15, wherein the at leastone first 5-position modified pyrimidine is a NapdC and the at least onesecond 5-position modified pyrimidine is selected from NapdU, 2NapdU,PPdU, MOEdU, TyrdU, TrpdU, and ThrdU.

Embodiment 27. The composition of embodiment 15, wherein the at leastone first 5-position modified pyrimidine is a PPdC and the at least onesecond 5-position modified pyrimidine is selected from NapdU, 2NapdU,PPdU, MOEdU, TrydU, TrpdU, and ThrdU.

Embodiment 28. The composition of embodiment 26 or embodiment 27,wherein the at least one second 5-position modified pyrimidine is aTyrdU.

Embodiment 29. The composition of any one of embodiments 15 to 28,wherein each polynucleotide comprises a random region.

Embodiment 30. The composition of embodiment 29, wherein the randomregion is 20 to 100, or 20 to 90, or 20 to 80, or 20 to 70, or 20 to 60,or 20 to 50, or 20 to 40, or 30 to 100, or 30 to 90, or 30 to 70, or 30to 60, or 30 to 50, or 30 to 40 nucleotides in length.

Embodiment 31. The composition of any one of embodiments 15 to 29,wherein each polynucleotide is 20 to 100, or 20 to 90, or 20 to 80, or20 to 70, or 20 to 60, or 20 to 50, or 30 to 100, or 30 to 90, or 30 to80, or 30 to 70, or 30 to 60, or 30 to 50, or 40 to 100, or 40 to 90, or40 to 80, or 40 to 70, or 40 to 60, or 40 to 50 nucleotides in length.

Embodiment 32. A composition comprising a first aptamer, a secondaptamer, and a target,

-   -   wherein the first aptamer comprises at least one first        5-position modified pyrimidine and at least one second        5-position modified pyrimidine;    -   wherein the second aptamer comprises at least one third        5-position modified pyrimidine;    -   wherein the first aptamer, second aptamer and the target are        capable of forming a trimer complex; and    -   wherein the first 5-position modified pyrimidine and the second        5-position modified pyrimidine are different 5-position modified        pyrimidines.

Embodiment 33. The composition of embodiment 32, wherein the first5-position modified pyrimidine is a 5-position modified uridine andwherein the second 5-position modified pyrimidine is a 5-positionmodified cytidine.

Embodiment 34. The composition of embodiment 32, wherein the first5-position modified pyrimidine is a 5-position modified cytidine andwherein the second 5-position modified pyrimidine is a 5-positionmodified uridine.

Embodiment 35. The composition of embodiment 33 or embodiment 34,wherein the 5-position modified uridine comprises a moiety at the5-position selected from a naphthyl moiety, a benzyl moiety, a tyrosylmoiety, an indole moiety and a morpholino moiety.

Embodiment 36. The composition of any one of embodiments 33 to 35,wherein the 5-position modified cytidine comprises a moiety at the5-position selected from a naphthyl moiety, a benzyl moiety, a tyrosylmoiety, and a morpholino moiety.

Embodiment 37. The composition of any one of embodiments 33 to 36,wherein the 5-position modified cytidine is selected from NapdC, 2NapdC,TyrdC, and PPdC.

Embodiment 38. The composition of any one of embodiments 33 to 37,wherein the 5-position modified uridine is selected from NapdU, 2NapdU,PPdU, MOEdU, TyrdU, TrpdU, and ThrdU.

Embodiment 39. The composition of embodiment 32, wherein the at leastone first 5-position modified pyrimidine is a NapdC and the at least onesecond 5-position modified pyrimidine is selected from NapdU, 2NapdU,PPdU, MOEdU, TyrdU, TrpdU, and ThrdU.

Embodiment 40. The composition of embodiment 32, wherein the at leastone first 5-position modified pyrimidine is a PPdC and the at least onesecond 5-position modified pyrimidine is selected from NapdU, 2NapdU,PPdU, MOEdU, TyrdU, TrpdU, and ThrdU.

Embodiment 41. The composition of embodiment 39 or embodiment 40,wherein the at least one second 5-position modified pyrimidine is aTyrdU.

Embodiment 42. The composition of any one of embodiments 32 to 41,wherein the third 5-position modified pyrimidine is selected from a5-position modified cytidine and a 5-position modified pyrimidine.

Embodiment 43. The composition of embodiment 42, wherein the third5-position modified pyrimidine is selected from BndC, PEdC, PPdC, NapdC,2NapdC, NEdC, 2NEdC, TyrdC, BndU, NapdU, PEdU, IbdU, FBndU, 2NapdU,NEdU, MBndU, BFdU, BTdU, PPdU, MOEdU, TyrdU, TrpdU, and ThrdU.

Embodiment 44. The composition of any one of embodiments 32 to 43,wherein the target is selected from a protein, a peptide, acarbohydrate, a small molecule, a cell and a tissue.

Embodiment 45. A method comprising:

-   -   (a) contacting an aptamer capable of binding to a target        molecule with a sample;    -   (b) incubating the aptamer with the sample to allow an        aptamer-target complex to form;    -   (c) enriching for the aptamer-target complex in the sample and    -   (c) detecting for the presence of the aptamer, aptamer-target        complex or target molecule, wherein the detection of the        aptamer, aptamer-target complex or target molecule indicates        that the target molecule is present in the sample, and wherein        the lack of detection of the aptamer, aptamer-target complex or        target molecule indicates that the target molecule is not        present in the sample;    -   wherein the aptamer is an aptamer of any one of embodiments 1 to        14.

Embodiment 46. The method of embodiment 45, wherein the method comprisesat least one additional step selected from: adding a competitor moleculeto the sample; capturing the aptamer-target complex on a solid support;and adding a competitor molecule and diluting the sample; wherein the atleast one additional step occurs after step (a) or step (b).

Embodiment 47. The method of embodiment 46, wherein the competitormolecule is selected from a polyanionic competitor.

Embodiment 48. The method of embodiment 47, wherein the polyanioniccompetitor is selected from an oligonucleotide, polydextran, DNA,heparin and dNTPs.

Embodiment 49. The method of embodiment 48, wherein polydextran isdextran sulfate; and DNA is herring sperm DNA or salmon sperm DNA.

Embodiment 50. The method of any one of embodiments 45 to 49, whereinthe target molecule is selected from a protein, a peptide, acarbohydrate, a small molecule, a cell and a tissue.

Embodiment 51. The method of any one of embodiments 45 to 50, whereinthe sample is selected from whole blood, leukocytes, peripheral bloodmononuclear cells, plasma, serum, sputum, breath, urine, semen, saliva,meningial fluid, amniotic fluid, glandular fluid, lymph fluid, nippleaspirate, bronchial aspirate, synovial fluid, joint aspirate, cells, acellular extract, stool, tissue, a tissue biopsy, and cerebrospinalfluid.

Embodiment 52. A method for detecting a target in a sample comprising

-   -   a) contacting the sample with a first aptamer to form a mixture,        wherein the first aptamer is capable of binding to the target to        form a first complex;    -   b) incubating the mixture under conditions that allow for the        first complex to form;    -   c) contacting the mixture with a second aptamer, wherein the        second aptamer is capable of binding the first complex to form a        second complex;    -   d) incubating the mixture under conditions that allow for the        second complex to form;    -   e) detecting for the presence or absence of the first aptamer,        the second aptamer, the target, the first complex or the second        complex in the mixture, wherein the presence of the first        aptamer, the second aptamer, the target, the first complex or        the second complex indicates that the target is present in the        sample;    -   wherein the first aptamer comprises at least one first        5-position modified pyrimidine and at least one second        5-position modified pyrimidine;    -   wherein the second aptamer comprises at least one third        5-position modified pyrimidine;    -   wherein the first 5-position modified pyrimidine and the second        5-position modified pyrimidine are different 5-position modified        pyrimidines.

Embodiment 53. The method of embodiment 52, wherein the first 5-positionmodified pyrimidine is a 5-position modified uridine and wherein thesecond 5-position modified pyrimidine is a 5-position modified cytidine.

Embodiment 54. The method of embodiment 53, wherein the first 5-positionmodified pyrimidine is a 5-position modified cytidine and wherein thesecond 5-position modified pyrimidine is a 5-position modified uridine.

Embodiment 55. The method of embodiment 53 or embodiment 54, wherein the5-position modified uridine comprises a moiety at the 5-positionselected from a naphthyl moiety, a benzyl moiety, a tyrosyl moiety, anindole moiety and a morpholino moiety.

Embodiment 56. The method of any one of embodiments 53 to 55, whereinthe 5-position modified cytidine comprises a moiety at the 5-positionselected from a naphthyl moiety, a benzyl moiety, a tyrosyl moiety, anda morpholino moiety.

Embodiment 57. The method of any one of embodiments 53 to 56, whereinthe 5-position modified cytidine is selected from NapdC, 2NapdC, TyrdC,and PPdC.

Embodiment 58. The method of any one of embodiments 53 to 57, whereinthe 5-position modified uridine is selected from NapdU, 2NapdU, PPdU,MOEdU, TyrdU, TrpdU, and ThrdU.

Embodiment 59. The method of embodiment 52, wherein the at least onefirst 5-position modified pyrimidine is a NapdC and the at least onesecond 5-position modified pyrimidine is selected from NapdU, 2NapdU,PPdU, MOEdU, TyrdU, TrpdU, and ThrdU.

Embodiment 60. The method of embodiment 52, wherein the at least onefirst 5-position modified pyrimidine is a PPdC and the at least onesecond 5-position modified pyrimidine is selected from NapdU, 2NapdU,PPdU, MOEdU, TyrdU, TrpdU, and ThrdU.

Embodiment 61. The method of embodiment 59 or embodiment 60, wherein theat least one second 5-position modified pyrimidine is a TyrdU.

Embodiment 62. The method of any one of embodiments 52 to 61, whereinthe third 5-position modified pyrimidine is selected from a 5-positionmodified cytidine and a 5-position modified pyrimidine.

Embodiment 63. The method of embodiment 62, wherein the third 5-positionmodified pyrimidine is selected from BndC, PEdC, PPdC, NapdC, 2NapdC,NEdC, 2NEdC, TyrdC, BNdU, NapdU, PedU, IbdU, FbndU, 2NapdU, NedU, MbndU,BfdU, BtdU, PpdU, MOEdU, TyrdU, TrpdU, and ThrdU.

Embodiment 64. The method of any one of embodiments 52 to 63, whereinthe target molecule is selected from a protein, a peptide, acarbohydrate, a small molecule, a cell and a tissue.

Embodiment 65. The method of any one of embodiments 52 to 64, whereinthe first aptamer, second aptamer and the target are capable of forminga trimer complex.

Embodiment 66. A method for identifying one or more aptamers capable ofbinding to a target molecule comprising:

-   -   (a) contacting a library of aptamers with the target molecule to        form a mixture, and allowing for the formation of an        aptamer-target complex, wherein the aptamer-target complex forms        when an aptamer has affinity for the target molecule;    -   (b) partitioning the aptamer-target complex from the remainder        of the mixture (or enriching for the aptamer-target complex);    -   (c) dissociating the aptamer-target complex; and    -   (d) identifying the one or more aptamers capable of binding to        the target molecule;    -   wherein the library of aptamers comprises a plurality of        polynucleotides, wherein each polynucleotide comprises at least        one first 5-position modified pyrimidine and at least one second        5-position modified pyrimidine, wherein the first 5-position        modified pyrimidine and the second 5-position modified        pyrimidine are different 5-position modified pyrimidines.

Embodiment 67. The method of embodiment 66, wherein each polynucleotidecomprises a fixed region at the 5′ end of the polynucleotide.

Embodiment 68. The method of embodiment 67, wherein the fixed region atthe 5′ end of each polynucleotide is at least 10, at least 15, at least20, at least 25 or at least 30 nucleotides in length, or 5 to 30, 10 to30, 15 to 30, 5 to 20, or 10 to 20 nucleotides in length.

Embodiment 69. The method of any one of embodiments 66 to 68, whereineach polynucleotide comprises a fixed region at the 3′ end of thepolynucleotide.

Embodiment 70. The method of embodiment 69, wherein the fixed region atthe 3′ end of the polynucleotide is at least 10, at least 15, at least20, at least 25 or at least 30 nucleotides in length, or 5 to 30, 10 to30, 15 to 30, 5 to 20, or 10 to 20 nucleotides in length.

Embodiment 71. The method of any one of embodiments 66 to 70, whereinthe first 5-position modified pyrimidine is a 5-position modifieduridine and wherein the second 5-position modified pyrimidine is a5-position modified cytidine.

Embodiment 72. The method of any one of embodiments 66 to 71, whereinthe first 5-position modified pyrimidine is a 5-position modifiedcytidine and wherein the second 5-position modified pyrimidine is a5-position modified uridine.

Embodiment 73. The method of embodiment 71 or embodiment 72, wherein the5-position modified uridine comprises a moiety at the 5-positionselected from a naphthyl moiety, a benzyl moiety, a tyrosyl moiety, anindole moiety and a morpholino moiety.

Embodiment 74. The method of any one of embodiments 71 to 73, whereinthe 5-position modified cytidine comprises a moiety at the 5-positionselected from a naphthyl moiety, a benzyl moiety, a tyrosyl moiety and amorpholino moiety.

Embodiment 75. The method of any one of embodiments 71 to 74, whereinthe 5-position modified cytidine is selected from NapdC, 2NapdC, TyrdC,and PPdC.

Embodiment 76. The method of any one of embodiments 71 to 75, whereinthe 5-position modified uridine is selected from NapdU, PPdU, MOEdU,TyrdU, TrpdU, and ThrdU.

Embodiment 77. The method of embodiment 66, wherein the at least onefirst 5-position modified pyrimidine is a NapdC and the at least onesecond 5-position modified pyrimidine is selected from NapdU, 2NapdU,PPdU, MOEdU, TrydU, TrpdU and ThrdU.

Embodiment 78. The method of embodiment 66, wherein the at least onefirst 5-position modified pyrimidine is a PPdC and the at least onesecond 5-position modified pyrimidine is selected from NapdU, 2NapdU,PPdU, MOEdU, TyrdU, TrpdU, and ThrdU.

Embodiment 79. The method of embodiment 77 or embodiment 78, wherein theat least one second 5-position modified pyrimidine is a TyrdU.

Embodiment 80. The method of any one of embodiments 66 to 79, whereineach polynucleotide comprises a random region.

Embodiment 81. The method of embodiment 80, wherein the random region is20 to 100, or 20 to 90, or 20 to 80, or 20 to 70, or 20 to 60, or 20 to50, or 20 to 40, or 30 to 100, or 30 to 90, or 30 to 70, or 30 to 60, or30 to 50, or 30 to 40 nucleotides in length.

Embodiment 82. The method of any one of embodiments 66 to 81, whereineach polynucleotide is 20 to 100, or 20 to 90, or 20 to 80, or 20 to 70,or 20 to 60, or 20 to 50, or 30 to 100, or 30 to 90, or 30 to 80, or 30to 70, or 30 to 60, or 30 to 50, or 40 to 100, or 40 to 90, or 40 to 80,or 40 to 70, or 40 to 60, or 40 to 50 nucleotides in length.

Embodiment 83. The method of any one of embodiments 66 to 82, whereineach polynucleotide is an aptamer that binds a target, and wherein thelibrary comprises at least 1000 aptamers, wherein each aptamer comprisesa different nucleotide sequence.

Embodiment 84. The method of any one of embodiments 66 to 83, whereinsteps (a), (b) and/or (c) are repeated at least one time, two times,three times, four times, five times, six times, seven times, eighttimes, nine times or ten times.

Embodiment 85. The method of any one of embodiments 66 to 84, whereinthe one or more aptamers capable of binding to the target molecule areamplified.

Embodiment 86. The method of any one of embodiments 66 to 85, whereinthe mixture comprises a polyanionic competitor molecule.

Embodiment 87. The method of embodiment 86, wherein the polyanioniccompetitor is selected from an oligonucleotide, polydextran, DNA,heparin and dNTPs.

Embodiment 88. The method of embodiment 87, wherein polydextran isdextran sulfate; and DNA is herring sperm DNA or salmon sperm DNA.

Embodiment 89. The method of any one of embodiments 66 to 88, whereinthe target molecule is selected from a protein, a peptide, acarbohydrate, a small molecule, a cell and a tissue.

Embodiment 90. The aptamer of any one of embodiments 1 to 14, whereinthe first 5-position modified pyrimidine and the second 5-positionmodified pyrimidine are capable of being incorporated by a polymeraseenzyme.

Embodiment 91. The composition of any one of embodiments 15 to 44,wherein the first 5-position modified pyrimidine and the second5-position modified pyrimidine are capable of being incorporated by apolymerase enzyme.

Embodiment 92. The method of any one of embodiments 45 to 89, whereinthe first 5-position modified pyrimidine and the second 5-positionmodified pyrimidine are capable of being incorporated by a polymeraseenzyme.

Embodiment 93. The aptamer of any one of embodiments 1 to 14 and 90,wherein the aptamer has improved nuclease stability compared to anaptamer of the same length and nucleobase sequence that comprises anunmodified pyrimidine in place of each of the first 5-position modifiedpyrimidines or an unmodified pyrimidine in place of each of the second5-position modified pyrimidine.

Embodiment 94. The aptamer of any one of embodiments 1 to 14, 90, and93, wherein the aptamer has a longer half-life in human serum comparedto an aptamer of the same length and nucleobase sequence that comprisesan unmodified pyrimidine in place of each of the first 5-positionmodified pyrimidines or an unmodified pyrimidine in place of each of thesecond 5-position modified pyrimidine.

EXAMPLES

The following examples are presented in order to more fully illustratesome embodiments of the invention. They should, in no way be construed,however, as limiting the broad scope of the invention. Those of ordinaryskill in the art can readily adopt the underlying principles of thisdiscovery to design various compounds without departing from the spiritof the current invention.

Example 1: Aptamers Comprising Two Modified Bases

To compare the relative efficiency of SELEX with two modified bases,five single modifications on dU (Nap-dU, PP-dU, MOE-dU, Tyr-dU andThr-dU) with unmodified dT as a control, and combinations withmodifications on dC (Nap-dC and PP-dC) with unmodified dC as a control,were analyzed, for a total of 18 starting libraries (FIG. 1 ). The typesof modifications tested included hydrophobic aromatic side chainsanalogous to hydrophobic side chains on amino acids. Hydrophilic sidechains on dU (MOE-dU and Thr-dU) were also tested. Each of the 18libraries contained 30 randomized nucleotides, allowing for ≥10¹⁵different sequences. The libraries were enzymatically synthesized usingnatural and/or modified nucleotide triphosphates using KOD DNApolymerase, Exo- (data not shown).

Thirty nucleotide (30N) randomized libraries were used instead of theprevious 40N randomized libraries with single modified dUs. Withoutintending to be bound by any particular theory, it was postulated thatincreasing the density of modified bases would allow for shorter highaffinity aptamers. Further, shorter oligonucleotide libraries givehigher yields. The ratio of each nucleotide was 1:1:1:1 for dA/dC/dG/dT(25% each). In each case, the random region was flanked with fixedsequences for hybridizing PCR amplification primers (Table 2), withadditional spacers at the 5′ end and at the 3′ end. The master synthetictemplate was used to generate modified libraries with all dU and or dCpositions uniformly modified in replacement primer extension reactions.

A total of 18 enzymatically synthesized libraries comprising singlemodified dU (Nap-dU, PP-dU, MOE-dU, Tyr-dU and/Thr-dU) with unmodifieddT as a control; single modified dC (Nap-dC and PP-dC) with unmodifieddC as a control; and combination of two modified bases: either Nap-dC orPP-dC, with all possible modified dUs (Nap-dU, PP-dU, MOE-dU, Tyr-dU andThr-dU). The qualitative primer extension reactions (in triplicates)were carried out using antisense template, radio-labeled 5′ primer withnatural or modified nucleotide tri-phosphates and KOD polymerase (Exo-)in solution, as follows. In a 60 μL primer extension reaction, 20 pmolesof biotinylated anti-sense library was mixed with 40 pmoles of 5′ coldprimer (2×) and trace amounts of ³²P labeled 5′ primer, 0.5 mM naturalor modified dNTP in 1×SQ20 buffer (120 mM Tris-HCl, pH 7.8; 10 mM KCl; 6mM (NH₄)₂SO₄; 7 mM MgSO₄, 0.1% Triton X-100 and 0.1 mg/mL BSA) and 0.25U/mL KOD Polymerase (Exo-). The mixture was heat cooled before addingDNA polymerase and the reaction was carried out at 68° C. for 2 hr, thencooled at 10° C. The fraction from each of the library reactions wereran on 10% TBU Urea gel along with free labeled primer. Small aliquotwas run on denaturing gels which were exposed to phosphor screens andimaged with Fuji phosphorimager, bands were quantitated using ImageGauge4.0 software and results were plotted in Graph pad Prism software 6.05.For making initial libraries large scale primer extension reactions werecarried out using master biotinylated antisense random library capturedon Pierce™ High Capacity Streptavidin Agarose beads (Life Technologies).Lower library yields were obtained for certain two modified nucleosidecombinations, for example, 28±1.3% for Nap-dC/Nap-dU, 40±5.2% forNap-dC/MOE-dU, and 43±2.7% for PP-dC/Nap-dU, compared to 100% unmodifiedDNA (dC/dT) library control. The frequency of each nucleotide wascalculated from the sequencing results obtained from the initial libraryand the master antisense random template used to generate each of thesingle and two base modified libraries. The master random antisensetemplate (30N) was chemically synthesized with 1:1:1:1 ratio ofdA:dG:dC:dT (TriLink Biotechnologies) at 1 μM scale. The initial randomsingle base and two base modified libraries were enzymaticallysynthesized in large scale reaction and used in selection experiments.These libraries were sequenced along with the enriched pools andnucleotide frequencies were plotted with total 100% for all four basesin 30N random region. No significant bias was observed in the nucleotidefrequencies when base composition of libraries was determined using deepsequencing compared with starting synthetic natural DNA template libraryand enzymatically synthesized unmodified DNA control initial library(data not shown).

The libraries were used to select aptamers that bind PCSK9. Theselections were carried out substantially as reported previously usingdextran sulphate as polyanionic competitor for a total of six roundsapplying incremental target dilution during each successive rounds ofselection. See Table 1 (R1=round 1, R2=round 2, etc.). Selection wasstarted by mixing modified random libraries (or control unmodified)(≥1000 pmol) and a human recombinant His-tagged target protein, PCSK9,which was present at 0.5 concentration, in 100 μL volume. The selectedcomplexes were partitioned on magnetic His-tag capture Dynabeads®,unbound sequences were washed, selected aptamers were eluted and PCRamplified using all natural nucleotides and 3′ biotin-primer. Thenatural double-stranded DNA biotinylated at 3′ end was captured onDynabeads® MyOne™ Streptavidin C1 beads, sense strands eliminated byalkali denaturation and replaced with modified dC and or dU in primerextension reactions to regenerate enriched pool and selection cycle wasrepeated with diluted protein. The concentration of protein for the nextround of SELEX was determined based on signal to background ratiocalculated from critical cycle time (Ct) value for each sample.

TABLE 1 In vitro selection conditions Library R1 DNA R2-R6 DNA PCSK9[nM] composition [nM] [nM] R1 R2 R3 R4 R5 R6 dC/dT (Control 10,000 100500 100 50 50 50 50 DNA) Nap-dC/dT 10,000 100 500 100 100 10 10 0.1PP-dC/dT 10,000 100 500 100 100 100 100 10 dC/Nap-dU 10,000 100 500 10050 50 5 0.5 dC/PP-dU 10,000 100 500 100 100 100 10 1 dC/MOE-dU 10,000100 500 100 100 100 100 100 dC/Tyr-dU 10,000 100 500 100 100 100 50 5dC/Thr-dU 10,000 100 500 100 100 100 100 10 Nap-dC/Nap-dU 10,000 100 500100 50 10 1 0.1 Nap-dC/PP-dU 10,000 100 500 100 100 10 1 0.1Nap-dC/MOE-dU 10,000 100 500 100 100 100 10 1 Nap-dC/Tyr-dU 10,000 100500 100 100 10 1 0.1 Nap-dC/Thr-dU 10,000 100 500 100 100 100 10 1PP-dC/PP-dU 10,000 100 500 100 100 100 10 1 PP-dC/Nap-dU 10,000 100 500100 100 100 10 1 PP-dC/MOE-dU 10,000 100 500 100 100 100 100 100PP-dC/Tyr-dU 10,000 100 500 100 50 10 1 0.1 PP-dC/Thr-dU 10,000 100 500100 100 100 10 1

The 5′ primer for amplification comprised a (AT4)-tail and the 3′ primercomprised a (A-biotin)2-T8-tail (SEQ ID NO: 82), which avoids additionof modified dC or dU when modified libraries are synthesized.

TABLE 2 Sequence of natural DNA template, primers used in SELEX NameSequence Anti-AB2- (Ab2)TTTTTTTTCTCTTTCTCTTCTCT 30N41.36CTTTCTCC30NGACCCACCCAGCGTGG SEQ ID NO: 1 (AT)4-5P41ATATATATCCACGCTGGGTGGGTC SEQ ID NO: 2 (AB)2(T)8-3P3(Ab2)TTTTTTTTCTCTTTCTCTTCTCT SEQ ID NO: 3 CTTTCTCC

After six rounds of selections, aptamers containing natural nucleotideswere deep sequenced using Ion Torrent PGM instrument. Sequence analysiswas performed using custom software using local batch alignment. Thedata from sequence analysis of all the enriched pools demonstrated thatthe two modified library combinations resulted in higher diversity inenriched sequences compared with single modified libraries (data notshown). To test binding affinity of aptamers, an extensive set ofsequences was chose representing not just high copy unique sequences butalso low copy sequences from distinct families (data not shown). All theaptamers were chemically synthesized by standard solid phasephosphoramidite chemistry using modified/unmodified phosphoramiditesreagents. All aptamers were initially screened as truncated 40merscontaining 30 nucleotide random region and additional 5 nucleotides fromfixed primer regions from 5′ and 3′ ends for their PCSK9 bindingaffinities in solution with radio-labeled filter-binding assays. Thetruncated aptamers (40mers) comprised 10 nucleotides from the fixedregions. The unmodified control DNA library (dC/dT) did not result inany active sequences (K_(d)<32 nM), which was expected as pool affinityfor this library was flat (data not shown) and also deep sequencing datadid not yield any enriched multi-copy sequences (data not shown). Thesingle modified libraries, with Nap (naphthyl) modification either on dCor dU resulted in aptamers having affinity for the target, however, theaptamers having the greatest affinity for the target were obtained withNap (naphthyl moiety) or PP (benzyl moiety) modified dC with Tyr(tyrosyl moiety) modified dU (FIG. 2 ). The replacement of Tyr-dU's withdT's abolished binding to the target, which indicates importance oftyrosyl moieties for binding interactions to target surface of PCSK9(data not shown). The affinity data demonstrated that two modifiednucleotide aptamers, in general, had greater affinity than singlemodified nucleotide aptamers, and also provided a greater number ofaptamers than bound to PCSK9 when compared with single modifiednucleotide aptamers (FIG. 3 ). Further, high copy single modifiednucleotide aptamers have average affinities between 0.1-100 nM, whilehigh copy two modified nucleotide aptamers have average affinities ≤0.1nM.

A summary of the data comparing single modified aptamers (40-mers) anddual-modified aptamers (40-mers) for PCSK9 is shown in the table 3below.

TABLE 3 Binding Data Summary for Single and Dual Modified Aptamers forPCSK9 PCSK9 Target % of Kd for Aptamer Total # of % of 5-PositionAptamers Total # of with Greatest Aptamers Aptamers Modification Testedwith a Aptamers Affinity for with no with no Category Of Aptamer Kd ≤10nM Tested Target Binding Binding Control dC/dT  0% 19 N/A 19 100%(unmodified) Single Mod. NapdC/dT 57% 23 0.25 nM 6  26% PPdC/dT  0% 8N/A 8 100% dC/NapdU 29% 24 0.28 nM 11  46% dC/PPdU 22% 18 0.18 nM 12 67% dC/MOEdU  0% 7 N/A 7 100% dC/TyrdU  0% 15   20 nM 9  60% dC/ThrdU 0% 18 N/A 18 100% Two Mod. NapdC/NapdU 70% 37 0.16 nM 7  19% NapdC/PPdU72% 32 0.05 nM 7  22% NapdC/MOEdU 20% 25 0.19 nM 14  56% NapdC/TyrdU 65%34 0.03 nM 10  29% NapdC/ThrdU  3% 40 0.23 nM 38  95% PPdC/PPdU 44% 340.13 nM 9  26% PPdC/NapdU 78% 32 0.14 nM 5  16% PPdC/MOEdU 17% 6  0.1 nM5  83% PPdC/TyrdU 80% 35 0.04 nM 5  14% PPdC/ThrdU  0% 35 N/A 35 100%

Based on the information in table 3, the percent of all single modifiedaptamers assayed that showed no binding was 62%. No binding is definedas an aptamer having a Kd of 320 nM or greater. The percent of allsingle modified aptamers with a Kd≤10 nM was less than 21%, and theaverage Kd for all single modified aptamers was 5.2 nM. In contrast, thepercent of all two modified (dual mod.) aptamers assayed that showed nobinding was 43%. Further, the percent of all two modified aptamers witha Kd≤10 nM was 47%, and the average Kd for all two modified aptamers was0.12 nM.

Example 2: Truncation of Dual-Modified Aptamers

The effect of further truncation on high-affinity (K_(d)<1 nM) aptamerbinding was investigated. Aptamers were truncated to 30mers, which is a25% reduction in length. The PP-dC/Tyr-dU combination had the highestnumber of aptamers that could be truncated to 30mers, while stillretaining binding affinity (FIG. 4A). Single base modified aptamersshowed truncability of 21.5% (blue bar, 3/14), two base modified Nap-dCaptamers with modified dU's showed truncability of ˜23% (red bar,11/48), while two base modified PP-dC with other modified dU's showedenhanced truncability of 60% (green bar, 27/45). The percentage and thenumber of 40mers that could be truncated to 30mers were also higher fortwo base modified combinations of PP-dC with PP-dU, Nap-dU, or Tyr-dUcompared with other libraries (FIG. 4B). Fewer aptamers from single basemodified libraries were tested because there were only 14 aptamers withaffinity ≤1 nM from the three libraries (40mers, in gray area in FIG. 4Bfor single mod). In contrast, the number of aptamers from two basemodified libraries with high affinity was 93 (40mers, in gray area ofFIG. 4B for two mod), 48 for Nap-dC with modified dU's and 45 for PP-dCwith modified dU's. Black horizontal line on each of the librariesindicates median value for the all aptamers in that library. Withoutintending to be bound by any particular theory, it is possible thatextended carbon chain in the PP modified base (compared with othermodifications) helps to reach inaccessible epitopes on the targetsurface and does not need fixed primer regions for the structuralfolding and effective protein binding interactions.

The specificity of PCSK9 aptamers to various other proproteinconvertases (PCs) was also evaluated. The three highest affinityaptamers from each library were selected (n=33, 40mers; none fromunmodified DNA control library, only two aptamers from dC/Tyr-dU libraryand one aptamer from PP-dC/MOE-dU library) and tested for theirspecificity to other PCs. The results demonstrated that the aptamerswere specific to PCSK9 and no detectable binding was observed with otherPCs (FIG. 5 ) at 100 nM concentrations.

The cross-species reactivity of truncated aptamers (n=41, 30mers) withK_(d) values of ≤1 nM was tested for rodent (mouse and rat) and Rhesusmonkey PCSK9 (see Table 4). The percent identity between PCSK9 fromvarious species is shown at the top of the graph in FIG. 6 . Themouse/rat PCSK9 is about 76% identical with monkey and human proteins.Most of the aptamers bound to Rhesus monkey PCSK9 with similaraffinities (identity 96.4%), however, few aptamers from two modifiedlibraries (PP-dC/Nap-dU and PP-dC/Tyr-dU) bound to rat and mouse PCSK9(identity ˜76%). These results demonstrated that certain two basemodified libraries (e.g., PP-dC/Nap-dU and PP-dC/Tyr-dU) generatedaptamers that can bind to both rodent and human/monkey PCSK9 withsimilar affinities (FIG. 6 ).

TABLE 4 Cross-species binding activity of single and double modifiedaptamers Human Monkey Mouse Rat PCSK9 PCSK9 PCSK9 PCSK9 Library Kd (pM)Kd (pM) Kd (pM) Kd (pM) Nap-dC/dT 150 149 — — Nap-dC/dT 114 56 — —dC/Nap-dU 223 174 — — Nap-dC/Nap-dU 362 461 — — Nap-dC/Nap-dU 609 1710 —— Nap-dC/Nap-dU 144 351 — — Nap-dC/Pp-dU 379 934 — — Nap-dC/Pp-dU 68 924— — Nap-dC/Moe-dU 82 83 — — Nap-dC/Moe-dU 172 169 — — Nap-dC/Tyr-dU 411177 — — Nap-dC/Tyr-dU 484 70 — — Nap-dC/Tyr-dU 191 193 — — Nap-dC/Tyr-dU805 987 — — Pp-dC/Pp-dU 186 189 — — Pp-dC/Pp-dU 162 198 — — Pp-dC/Pp-dU220 1150 — — Pp-dC/Pp-dU 564 638 — — Pp-dC/Pp-dU 53 106 — — Pp-dC/Pp-dU213 229 — — Pp-dC/Pp-dU 378 306 — — Pp-dC/Pp-dU 93 204 — — Pp-dC/Pp-dU413 402 — — Pp-dC/Nap-dU 283 247 23800 — Pp-dC/Nap-dU 70 74 742 1440Pp-dC/Nap-dU 69 117 — — Pp-dC/Nap-dU 111 140 500 680 Pp-dC/Nap-dU 136205 3820 18100 Pp-dC/Nap-dU 800 780 — — Pp-dC/Nap-dU 127 128 1950 45000Pp-dC/Tyr-dU 701 — — — Pp-dC/Tyr-dU 187 109 — — Pp-dC/Tyr-dU 93 — — —Pp-dC/Tyr-dU 61 45 — — Pp-dC/Tyr-dU 62 — — — Pp-dC/Tyr-dU 451 350 1090858 Pp-dC/Tyr-dU 12 21 — — Pp-dC/Tyr-dU 122 — — — Pp-dC/Tyr-dU 28 — — —Pp-dC/Tyr-dU 148 — — — Pp-dC/Tyr-dU 109 — — — “—” indicates that nobinding was detected in the assay

Example 3: Aptamer Binding in Sandwich Assays

SELEX method sometimes yields aptamers that preferentially bind to adominant “aptagenic” epitopes on the target surface. Therefore, reportson aptamer sandwich pairs are limited in the literature. Modificationsin the selection method may be employed to search for the aptamers thatcan bind to different epitopes on the target protein, such asmultivalent aptamer isolation (MAI-SELEX), array-based discoveryplatform for multivalent aptamer (AD-MAP) sandwich selections, in whichprimary aptamer is used in excess to block the first epitope in aneffort to discover second aptamer binding to a non-competing signalingepitope. To demonstrate if expanded chemical diversity generated bymultiplicity in the modifications on dC and dU together in selectingaptamers that can bind to different epitopes on the target surface,bead-based sandwich pair screening assays were developed in whichLuminex® MagPlex® avidin coupled magnetic beads were used to capturebiotinylated primary aptamer (FIG. 7A). The capture beads withindividual aptamers were used mixed together to search for secondbinding partner in a multiplex pair-wise combination (FIG. 7A). For thisexperiment, 40mer aptamers (n=96, 9216 pairs) with affinity K_(d)≤1 nMfrom single and two base modified libraries were used. Briefly,individual aptamers (0.05 pmoles per sample) with were captured onsingle MagPlex Avidin bead type and mixed together (24 beads in oneexperiment, 1000 beads per sample) and captured for 20 min at roomtemperature with shaking at 1850 rpm. Beads were washed with 1×SBT for 2min followed with 0.5 mM free biotin wash for 5 min in 1×SBT, followedby 3 washes of 1×SBT for 2 min each. Beads were blocked with freeStreptavidin for 5 min and washed again for 2 min with 1×SBT. The 24different bead types with individual aptamers were mixed together forscreening of sandwich partner for each of the capture aptamer. Adetection or secondary aptamer was diluted to 500 nM in 1×SBT,heat-cooled and mixed with PCSK9 (final 10 nM), incubated at 25° C. for1 hr. 1000 capture beads were added and incubated further for 1 hr withshaking. Beads were then captured on a magnet, washed three times with1×SBT for 2 min each and re-suspended in 75 μL 1×SBT with 0.1% BSA and100 uM DxSO4. To this 75 μL of Streptavidin phycoerythrin (final 5μg/mL) was added and incubated at 25° C. for 20 min with shaking. Beadswere finally washed again for 2 min with 1×SBT and read on Luminex 3DxMAP machine.

The single base modified libraries generated few sandwich pairs (three),while adding aptamers from two base modified libraries in combinationwith single base modified aptamers resulted in more sandwich pairs (22pairs). Moreover, the number of sandwich pairs per library wasdramatically increased when both partners (capture and detection)aptamers came from the two base modified libraries (45 pairs, FIG. 7C,FIG. 7B).

The multiple epitope binding results from the sandwich screeningsuggested that the increase in the chemical diversity in initial randomlibrary resulted in modified aptamers that can bind to non-competingsites on the target surface. Next, the sandwich pairs that resulted inhighest signals (10 nM PCSK9 concentration; 0.75% of all pairs tested,70 pairs out of 9216; FIG. 7C) were measured for PCSK9 concentrationdependent responses with a subset of results shown in FIGS. 8A (theconcentration dependent signals are shown for the single base modifiedprimary aptamer dC/PP-dU and the best secondary that worked well was thetwo base modified aptamer Nap-dC/Nap-dU [triangles]) and 8B (theconcentration dependent signals are shown for the two base modifiedsecondary aptamer Nap-dC/Nap-dU and the best primary that worked wellwas the single base modified aptamer dC/PP-dU [closed squares]).Interestingly, one specific pair, constituting a single base modifiedprimary (PP-dU, affinity, Kd of 175 pM) and a two base modifiedsecondary (Nap-dU/Nap-dC, affinity, Kd of 531 pM), resulted in therobust signal that was much higher than any other pairs in oneorientation (FIG. 8C). However, when this single base modified primaryaptamer was switched to a secondary aptamer, signal was lost, whichindicated that the orientation of the aptamers was important for thissandwich pair. This aptamer sandwich pair can also measure activity of again-of-function mutant protein, PCSK9 D374Y (FIG. 8D), which has higheraffinity for LDL-R than wild type PCSK9 and is reported to beover-expressed in patients with severe form of familialhypercholesterolemia (FH). The sensitivity and MFU values were higherfor the mutant PCSK9 D374Y than the wild type protein.

The specificity of an aptamer sandwich pair was also measured by lack ofendogenous signal when recombinant human PCSK9 was spiked into newborncalf serum (NBCS) compared to human plasma (data not shown).

This sandwich pair was further characterized to develop an aptamersandwich assay to detect circulating concentrations of plasma PCSK9 inhuman clinical samples. The performance of the sandwich assay wasevaluated by conducting studies such as sensitivity (FIGS. 9A and 9B andTables 5 and 6), precision (Tables 7 and 8), accuracy (Table 9) andplasma dilution linearity measurements (FIG. 10 ), all of whichconfirmed a robust assay window. To assess the sample dilution linearityof the assay, a sandwich assay was performed with samples containingand/or spiked with high concentrations of PCSK9. The plasma samples(n=5) were serially diluted with the assay buffer to fit the valueswithin the dynamic range of the assay.

The limit of detection (LLoD), defined as the concentration of PCSK9 (40pg/mL) giving an RFU value higher than the mean RFU of blank (dilutionbuffer) plus 3 standard deviations, is shown in Table 5. The lower limitof quantification (LLoQ) and upper limit of quantification (ULoQ),defined as lowest and highest concentrations of PCSK9 that can bequantitated using 4 parameter logistic (4PL) fit applied to the standardcurves resulting in 80-120% recovery of the known target concentrations,are shown in Table 6. To determine intra-assay variability, five plasmasamples of known concentrations were tested 16 times in single plate.See Table 7. Coefficients of variability (CVs) within the assay rangedfrom 4.3% to 6%. To determine inter-assay variability, five plasmasamples of known concentrations were measured in five separate assays.See Table 8. CVs between the assays ranged from 2.3% to 9.8%. Finally,to determine the accuracy in target measurement, five plasma sampleswere spiked with different amounts of PCSK9 and measured. The recoveryof spiked PCSK9 levels throughout the range of the assay was evaluated.See Table 9. The percent recovery of samples averaged from 83.1% to137.5% of the spiked target.

A set of plasma samples obtained from two groups of individuals, onecontrol group (n=42) and other study group in which subjects were onLipitor® statin therapy (n=42, by self-report) was evaluated in order todetermine if the assay can statistically differentiate between these twogroups, because it is known that use of statins increases plasmaconcentrations of PCSK9. The sandwich assay was developed using acapture or primary SOMAmer (11723-5) as a single base modified aptamer(PP-dU/dC) and a secondary or detection aptamer (11727-20) as a two basemodified aptamer (Nap-dC/Nap-dU).

These results indicated that aptamer sandwich assay can statisticallydifferentiate between the two groups with P value of 0.0044 (FIG. 11 )by Mann-Whitney analysis and that this assay could have use inidentifying people who could benefit from anti-PCSK9 therapy due totheir high plasma concentrations of PCSK9.

An aptamer sandwich assay was also used to measure PCSK9 concentrationsin cell-free supernatants from PCSK9 over-expressing HepG2 cells toidentify the over-expressing clones and to demonstrate the researchutility of the assay (FIG. 12 ). PCSK9 was over-expressed in HepG2 cellsusing the SBI System Biosciences LentiViral system (LV300A-1). The HepG2cell line was transduced with lentiviral expression clone for wild typehuman PCSK9 obtained from Origene (RC220000L1) for the generation ofstable cell line. A total of 96 individual clones were screened fortheir ability to secrete PCSK9. The relative amount of PCSK9 secreted inthe medium was measured by the aptamer sandwich assay for each clone andwas compared with expression from wild-type HepG2 cells. The number ofcells used to produce recombinant protein was normalized using the CellTiter-Glo® Luminescent cell viability assay. Clone number 45 secreted˜100-fold more PCSK9 than wild-type HepG2 cells.

TABLE 5 Sensitivity of sandwich assay (lower limit of detection) LLoDQuantitation PCSK9 No. Blank (40 pg/mL)  1 127.5 175.5  2 124 175.5  3122 179  4 129 181.5  5 136 189.5  6 125.5 185.5  7 117 174.5  8 127.5170  9 115.5 173.5 10 121 179.5 11 128 166 12 120.5 181.5 13 120.5 181.514 130.5 170.5 15 115.5 172 16 130 164 CV (%) 4.73 3.93

TABLE 6 Sensitivity of sandwich assay (lower limit of quantification)ULoQ Quantitation LLoQ Quantitation Logistic 4PL (R² = 0.99) Logistic4PL (R² = 0.99) PCSK9 % Std PCSK9 % Std (ng/mL) Recovery (ng/mL)Recovery Std 1 100 Out of range 5 100.1 Std 2 31.6000 72.3 2.5 99.3 Std3 9.9856 94.5 1.25 102.6 Std 4 3.1554 102.2 0.625 100.2 Std 5 0.9971112.1 0.3125 98.0 Std 6 0.3151 108.9 0.156 98.8 Std 7 0.0996 89.1 0.078105.5 Std 8 0.0315 54.6 0.039 79.9 Std 9 0.0099 Out of range 0.020 82.6Std 10 0.0031 Out of range 0.010 67.2 Std 11 0.0010 Out of range 0.005Out of range

TABLE 7 Precision within assays PCSK9 (ng/mL) No. Plasma 1 Plasma 2Plasma 3 Plasma 4 Plasma 5  1 139.4 118.8 196.1 220.8 104.4  2 149.4120.7 164.4 208.5 84.7  3 142 115.9 186.4 224.9 102.3  4 151.4 125.8196.7 227.1 102  5 147.7 125.6 193.9 226.3 96.7  6 146.6 125.3 183.2235.2 98.5  7 148.4 123.7 179.4 237.8 100.9  8 133.8 127.6 172 245.194.3  9 154.7 130.2 191.3 240.3 100.3 10 147 117.5 181.7 228.3 104.2 11133 121.3 190.6 229.5 89.3 12 150.9 127.9 189.8 231.3 98 13 146 121.1193.2 218.5 91.4 14 152.9 125.8 195.2 232.5 100.5 15 126.8 117.7 173.1220.4 88.2 16 153.6 109 172.9 235.6 93.8 CV (%) 5.4 4.3 5.3 3.9 6

TABLE 8 Precision between assays PCSK9 (ng/mL) No. Plasma 1 Plasma 2Plasma 3 Plasma 4 Plasma 5 Run 1 143.8 111.6 187.0 210.7 99.5 Run 2130.2 99.6 176.7 199.3 93.0 Run 3 117.3 106.2 164.4 200.8 83.2 Run 4141.7 111.8 192.2 206.1 90.7 Run 5 151.5 112.6 197.1 206.9 91.8 CV (%)9.8 5.1 7.1 2.3 6.4

TABLE 9 Accuracy of target measurement None +300 +100 +30 Sample(unspiked) (ng/mL) (ng/mL) (ng/mL) Plasma 1 PCSK9 92.4 359.0 192.2 133.7(ng/mL) Recovery — 88.9 99.8 137.5 (%) Plasma 2 PCSK9 76.9 351.7 179.8109.2 (ng/mL) Recovery — 91.6 102.9 107.6 (%) Plasma 3 PCSK9 137.4 454.5256.9 172.9 (ng/mL) Recovery — 105.7 119.5 118.3 (%) Plasma 4 PCSK9172.0 474.8 275.5 202.3 (ng/mL) Recovery — 100.9 103.5 101.1 (%) Plasma5 PCSK9 70.1 334.5 190.6 95.0 (ng/mL) Recovery — 88.1 120.5 83.1 (%)

Example 4: Target Activity Inhibition by Dual-Modified Aptamers

To find inhibitors of PCSK9 that block binding of PCSK9 to LDL-R, 41truncated 30mer aptamers with K_(d)≤1 nM were screened in a plate-basedassay in which plates were coated with LDL-R and binding of biotinylatedPCSK9 was detected using streptavidin-HRP conjugate by chemiluminescentreagents (FIG. 13 ). The recombinant LDL-R (Acro Biosystems) was coatedon the ELISA plates (2.5 μg/mL) overnight at 4° C., and then wells werewashed and blocked with Super Block (Invitrogen) for 1 hr at roomtemperature. The biotinylated PCSK9 (Acro Biosystems, Avi-tagged) andaptamer were mixed together and incubated at room temperature for 1 hr,then added to the ELISA plate and further incubated for 2 hrs at roomtemperature with shaking. The top PCSK9 concentration was 0.5 nM and thetop concentration for aptamer was 100 nM, and these were then seriallydiluted by ½ log for the inhibition curve. Streptavidin conjugated HRP(Invitrogen, 1 μg/ml) was added to the wells and incubated for 30 min atroom temperature with shaking, Pico-sensitive chemiluminescencesubstrates (Invitrogen) were added, luminescence was measured inLuminometer (Hidex Plate Chameleon), and data were plotted in Graph PadPrism 6.0 software to calculate the EC₅₀ values. A PCSK9 neutralizingantibody (BPS Bioscience) was used as the control.

The results from the inhibition assay (testing concentrations ofaptamers at 100 nM and PCSK9 at 1 nM) showed that 70% of the aptamerswere inhibitors (over 90% inhibition) and that some of the two modifiedaptamers potently inhibited PCSK9 interactions with LDL-R (data notshown). Aptamers were further evaluated for dose-response curves todetermine their EC₅₀ values for inhibition. The results indicated thatmany of inhibitors potently inhibited the PCSK9 interaction with LDL-Rwith an EC₅₀ in the 0.1-1 nM range (FIG. 14 ). To demonstrate potentialtherapeutic value in two modified aptamers, one species cross-reactivePCSK9 aptamer (30mer, Seq ID. 11733-91_5 (11733-198)) was chosen formeasurement of target affinity to PCSK9's from various species. Thisaptamer had affinity of 14.7, 11.3, 5.2, 77 and 165 pM to human(wild-type), rhesus monkey, human (GOF mutant D374Y), mouse, and ratPCSK9, respectively (FIG. 15A). This aptamer also blocked the wild-typehuman PCSK9 LDL-R interaction with an EC₅₀ of 2.1 nM and the GOF mutantPCSK9 D374Y LDL-R interaction with an EC₅₀ of 3.6 nM (FIG. 15B). Thespecificity of this aptamer for PCSK9 compared with other PCs wasevaluated, and results showed that this aptamer bound to only PCSK9 andnot to other PCs (data not shown).

To test the neutralizing effect of the PCSK9 aptamer in blocking LDLRdegradation, a PP-dC/Nap-dU aptamer was tested in an LDL uptake reversalassay in which wild-type HepG2 cells were incubated for 16 hrs withrecombinant PCSK9, and then cells were washed and fluorescently-labeledLDL was added for 3 hrs. The results showed that the aptamer can reverseLDL-uptake with an EC₅₀ of 159 nM (FIG. 16 and FIG. 17 ). Further,aptamer treatment can increase the LDL-R levels in PCSK9 over-expressingHepG2 cells as measured by FACS (FIG. 17 . These results with a speciescross-reactive, high-affinity, truncated, specific and highly potentaptamer suggest that the potential therapeutic value of a two basemodified aptamer could be further optimized for length and bio-stabilityby post-SELEX modification.

Example 5: Serum Stability of Dual-Modified Aptamers

To determine the serum-stability of the dual-modified aptamers in humanserum, 1 μM gel-purified aptamer was incubated in 90% pooled human serumin PBS buffer in a total volume of 200 μL at 37° C. At various timepoints, 20 μL aliquots were collected and an equal volume ofEDTA/formamide/dye mix (Formamide 87.7%, SDS, 0.03%, Sodium EDTA, 20 mM,Xylene Cyanol, 0.05%, Bromophenol Blue, 0.05%, Orange G, 0.1%) wasadded. The aliquot mixes were then stored at −20° C. Prior to analysis,the 40 μL aliquot mix was diluted with 100 μL H₂O and extracted with 150μL 25:24:1 phenol:chloroform:isoamyl alcohol. The samples werecentrifuged and 16,100×g for 15 minutes, and the aptamer-containingaqueous phase was removed and stored at −20° C. until gel analysis.

The aptamer samples were loaded on a 15% TBE PAGE denaturing gel (8 Murea), and the aptamer stained with 1× (˜2 μM) SYBR Gold for 10 minutes.The amount of full-length aptamer at each time point was quantifiedusing FluorChemQ analysis software (AlphaInnotech).

The results of that experiment are shown in Table 10 and FIG. 18 . Table10 shows the composition of the aptamers tested in that experiment, thepercentage of full-length aptamer remaining after 96 hours in 90% humanserum and the half-life of each aptamer, which was calculated by linearregression fit using gel quantified data in GraphPad Prism 7 software.FIG. 18 shows the percentage of full-length aptamer remaining over time.In general, dual modified aptamers demonstrated greater stability inhuman serum over time than the single modified aptamers.

TABLE 10 Aptamer composition. % FL at Half-Life Aptamer length dC mod dUmod # dC mod (%) # dU mod (%) # A (% A) # C (% C) # G (% G) # T (% T) 96hours (hours) dC/dT 29 none none none none  7 (24 %) 4 (14 %)  8 (28%)10 (34%) 0 11 NapdC/dT 30 Nap-dC none 12 (40%) none  4 (13.3%) none  9(30%)  5 (16.7%) 39 72 dC/NapdU 30 none Nap-dU none  5 (16.7%) 10(33.3%) 6 (20.0%)  9 (30%) none 17 23 dC/PPdU 30 none PP-dU none 11(36.7%)  7 (23.3%) 6 (20.0%)  6 (20.0%) none 57 53 NapdC/NapdU 30 Nap-dCNap-dU  5 (16.7%)  4 (13.3%)  9 (30%) none 12 (40%) none 65 101NapdC/PPdU 30 Nap-dC PP-dU  7 (23.3%)  8 (26.7%)  5 (16.7%) none 10(33.3%) none 44 53 NapdC/MOEdU 30 Nap-dC MOE-dU 12 (40%)  3 (10.0%)  6(20.0%) none  9 (30%) none 40 43 NapdC/TyrdU 30 Nap-dC Tyr-dU 11 (36.7%) 6 (20.0%)  3 (10.0%) none 10 (33.3%) none 104 690 PPdC/PPdU 30 PP-dCPP-dU  7 (23.3%)  8 (26.7%)  6 (20.0%) none  9 (30%) none 108 633PPdC/NapdU 30 PP-dC Nap-dU  7 (23.3%)  9 (30%)  6 (20.0%) none  8(26.7%) none 86 847 PPdC/TyrdU 30 PP-dC Tyr-dU  6 (20.0%) 12 (40%)  7(23.3%) none  5 (16.7%) none 75 907

Example 6: Aptamers Comprising Two Modified Bases

The libraries described in Example 1 were used to select aptamers thatbind to ErbB2, ErbB3, and PSMA. The selections were carried out for eachtarget substantially as described in Example 1. For ErbB2 and ErbB3, thesingle-modified Nap-dC/dT; PP-dC/dT; and dC/Tyr-dU libraries; and thedual-modified Nap-dC/Tyr-dU, and PP-dC/Tyr-dU libraries were used. ForPSMA, the unmodified dC/dT library; the single-modified Nap-dC/dT;PP-dC/dT; dC/Nap-dU, dC-PP-dU, dC-MOE-dU, and dC/Tyr-dU libraries; andthe dual-modified Nap-dC/Nap-dU, Nap-dC/PP-dU, Nap-dC/MOE-dU,Nap-dC/Tyr-dU, PP-dC/PP-dU, PP-dC/Nap-dU, and PP-dC/Tyr-dU librarieswere used.

As before, the unmodified control DNA library (dC/dT), which was usedfor PSMA, did not result in any aptamers that bound to PSMA. The singlemodified libraries, with Nap modification (naphthyl moiety) either on dCor dU resulted in binders for all three targets, however, thedual-modified libraries provided aptamers with greater affinity relativeto the single-modified libraries (FIG. 19A-C).

A summary of the data comparing the single modified aptamers (40-mers)and dual-modified aptamers (40-mers) for each of PSMA, ErbB2 and ErbB3are shown in tables 11, 12 and 13 respectively.

TABLE 11 Binding Data Summary for Single and Dual Modified Aptamers forPSMA PSMA Target % of Kd for Aptamer Total # of % of 5-Position AptamersTotal # of with Greatest Aptamers Aptamers Modification Tested with aAptamers Affinity for with no with no Category Of Aptamer Kd ≤10 nMTested Target Binding Binding Control dC/dT  0% 6 N/A 6 100%(unmodified) Single Mod. NapdC/dT 36% 25  0.5 nM 7  28% PPdC/dT  0% 7N/A 7 100% dC/NapdU 13% 24  6.7 nM 19  79% dC/PPdU  0% 20 35.5 nM 17 85% dC/MOEdU  0% 1 N/A 1 100% dC/TyrdU  5% 21  6.3 nM 19  90% dC/ThrdUN.T. N.T. N.T. N.T. N.T. Two Mod. NapdC/NapdU 12% 17   1 nM 9  53%NapdC/PPdU 12% 17  0.5 nM 9  53% NapdC/MOEdU 15% 13  1.1 nM 7  54%NapdC/TyrdU 58% 26  0.3 nM 9  35% NapdC/ThrdU N.T. N.T. N.T. N.T. N.T.PPdC/PPdU 15% 20  1.6 nM 8  40% PPdC/NapdU 18% 17   3 nM 10  59%PPdC/MOEdU N.T. N.T. N.T. N.T. N.T. PPdC/TyrdU  0% 24 38.9 nM 20  83%PPdC/ThrdU N.T. N.T. N.T. N.T. N.T. N.T. is “not tested”; N/A is notapplicable or no data

Based on the information in table 11, the percent of all single modifiedaptamers assayed that showed no binding was 71%. No binding is definedas an aptamer having a Kd of 320 nM or greater. The percent of allsingle modified aptamers with a Kd≤10 nM was 12%, and the average Kd forall single modified aptamers was 12.3 nM. In contrast, the percent ofall two modified (dual mod.) aptamers assayed that showed no binding was54%. Further, the percent of all two modified aptamers with a Kd≤10 nMwas 20%, and the average Kd for all two modified aptamers was 6.6 nM.

TABLE 12 Binding Data Summary for Single and Dual Modified Aptamers forERBB2 ERBB2 Target % of Kd for Aptamer Total # of % of 5-PositionAptamers Total # of with Greatest Aptamers Aptamers Modification Testedwith a Aptamers Affinity for with no with no Category Of Aptamer Kd ≤10nM Tested Target Binding Binding Control dC/dT N.T. N.T. N.T. N.T. N.T.(unmodified) Single Mod. NapdC/dT  0% 23 13.5 nM 12 52% PPdC/dT  7% 15 7.8 nM  8 53% dC/NapdU N.T. N.T. N.T. N.T. N.T. dC/PPdU N.T. N.T. N.T.N.T. N.T. dC/MOEdU N.T. N.T. N.T. N.T. N.T. dC/TyrdU  0% 29 24.1 nM 2793% dC/ThrdU N.T. N.T. N.T. N.T. N.T. Two Mod. NapdC/NapdU N.T. N.T.N.T. N.T. N.T. NapdC/PPdU N.T. N.T. N.T. N.T. N.T. NapdC/MOEdU N.T. N.T.N.T. N.T. N.T. NapdC/TyrdU 28% 32 0.65 nM 10 31% NapdC/ThrdU N.T. N.T.N.T. N.T. N.T. PPdC/PPdU N.T. N.T. N.T. N.T. N.T. PPdC/NapdU N.T. N.T.N.T. N.T. N.T. PPdC/MOEdU N.T. N.T. N.T. N.T. N.T. PPdC/TyrdU 20% 200.74 nM 13 65% PPdC/ThrdU N.T. N.T. N.T. N.T. N.T.

Based on the information in table 12, the percent of all single modifiedaptamers assayed that showed no binding was 70%. No binding is definedas an aptamer having a Kd of 320 nM or greater. The percent of allsingle modified aptamers with a Kd≤10 nM was less than 2%, and theaverage Kd for all single modified aptamers was 15.1 nM. In contrast,the percent of all two modified (dual mod.) aptamers assayed that showedno binding was 44%. Further, the percent of all two modified aptamerswith a Kd≤10 nM was 25%, and the average Kd for all two modifiedaptamers was 0.7 nM.

TABLE 13 Binding Data Summary for Single and Dual Modified Aptamers forERBB3 ERBB3 Target % of Kd for Aptamer Total # of % of 5-PositionAptamers Total # of with Greatest Aptamers Aptamers Modification Testedwith a Aptamers Affinity for with no with no Category Of Aptamer Kd ≤10nM Tested Target Binding Binding Control dC/dT N.T. N.T. N.T. N.T. N.T.(unmodified) NapdC/dT 75% 28 0.035 nM  5 18% PPdC/dT  0% 12  17.5 nM  325% dC/NapdU N.T. N.T. N.T. N.T. N.T. Single Mod. dC/PPdU N.T. N.T. N.T.N.T. N.T. dC/MOEdU N.T. N.T. N.T. N.T. N.T. dC/TyrdU 17% 23  0.35 nM 1670% dC/ThrdU N.T. N.T. N.T. N.T. N.T. NapdC/NapdU N.T. N.T. N.T. N.T.N.T. NapdC/PPdU N.T. N.T. N.T. N.T. N.T. NapdC/MOEdU N.T. N.T. N.T. N.T.N.T. NapdC/TyrdU 69% 39  0.02 nM 10 26% NapdC/ThrdU N.T. N.T. N.T. N.T.N.T. Two Mod. PPdC/PPdU N.T. N.T. N.T. N.T. N.T. PPdC/NapdU N.T. N.T.N.T. N.T. N.T. PPdC/MOEdU N.T. N.T. N.T. N.T. N.T. PPdC/TyrdU 69% 35 0.02 nM  8 23% PPdC/ThrdU N.T. N.T. N.T. N.T. N.T.

Based on the information in table 13, the percent of all single modifiedaptamers assayed that showed no binding was 38%. No binding is definedas an aptamer having a Kd of 320 nM or greater. The percent of allsingle modified aptamers with a Kd≤10 nM was 40%, and the average Kd forall single modified aptamers was 6 nM. In contrast, the percent of alltwo modified (dual mod.) aptamers assayed that showed no binding was24%. Further, the percent of all two modified aptamers with a Kd≤10 nMwas 69%, and the average Kd for all two modified aptamers was 0.02 nM.

Example 7: Further Aptamers Comprising Two Modified Bases

Libraries comprising each of the modification pairs shown in Table 14are made as follows. In some embodiments, each library contains 40 ormore randomized nucleotides. In some embodiments, each library contains30 randomized nucleotides, allowing for ≥10¹⁵ different sequences. Thelibraries may be enzymatically synthesized using natural and/or modifiednucleotide triphosphates using KOD DNA polymerase, Exo-. In someembodiments, the random region is flanked with fixed sequences forhybridizing PCR amplification primers, with or without additionalspacers at the 5′ end and at the 3′ end. In some instances, the mastersynthetic template is used to generate modified libraries with all dUand or dC positions uniformly modified in replacement primer extensionreactions. The library synthesis may be performed substantially asdescribed in Example 1.

TABLE 14 Dual modified aptamer libraries Library  1 dC/dT (DNA Control) 2 Nap-dC/dT  3 2Nap-dC/dT  4 PP-dC/dT  5 Tyr-dC/dT  6 dC/Nap-dU  7dC/2Nap-dU  8 dC/PPdU  9 dC/Trp-dU 10 dC/Tyr-dU 11 Nap-dC/Nap-dU 12Nap-dC/2Nap-dU 13 Nap-dC/PP-dU 14 Nap-dC/Trp-dU 15 Nap-dC/Tyr-dU 162Nap-dC/Nap-dU 17 2Nap-dC/2Nap-dU 18 2Nap-dC/PP-dU 19 2Nap-dC/Trp-dU 202Nap-dC/Tyr-dU 21 PP-dC/Nap-dU 22 PP-dC/2Nap-dU 23 PP-dC/PP-dU 24PP-dC/Trp-dU 25 PP-dC/Tyr-dU 26 Tyr-dC/Nap-dU 27 Tyr-dC/2Nap-dU 28Tyr-dC/PP-dU 29 Tyr-dC/Trp-dU 30 Tyr-dC/Tyr-dU

One or more of the libraries in Table 14 may be used to select aptamersthat bind to a target, such as a protein target. The librariescomprising two modified bases typically yield aptamers having greaterspecificity and/or affinity for the target.

Example 8: Exemplary Dual-Modified Aptamers

PCSK9-binding aptamers of the conserved sequence family from pool 11720(Nap-dC/dT) are shown in Table 15. Only the random region of eachsequence is shown. The number of copies of each sequence (identical orequivalent with up to 5 mismatches) out of 11,380 total sequences isindicated. All Aptamers in this family share the conserved sequenceelement TTppGGpp, where p=Nap-dC. Aptamer 11730-6 (SEQ ID No: 4, Kd=0.1nM) was the representative chosen from this pool for the metabolicstability assay.

TABLE 15 Aptamers from pool 11720 SEQ ID No CopiesRandom Region Sequence 4 401               AAG TTppGGppGppTpGGGGTpppTGppAA 5 21        ATAppTGGGA TTppGGpp ATTTGpGpAGTT 6 4            TGAAG TTppGGpp GTGpGpATGGTApppAT 7 3           TTTGTGpTTppGGpp TAGpGpAGATATppT 8 1             ATAGG TTppGGpp TTGpGpTGTTTAGApA9 1    TAGATGppTGGTAT TTppGGpp TTGpGpAT 10 2   TAGTGpppTGATpTA TTppGGppAAGpppA 11 3  TTTGppppTGGTTApG TTppGGpp TGGpGpA 12 2            ATGppGTTppGGpp TAGpGpTpGTTApppA 13 1   TGAppAppTGTppAA TTppGGpp TAGpGpA 14 1        TAppAGGTA TTppGGpp GAGpGpTGpTATA 15 1      GAGpppAGTTAp TTppGGppTTGpATTGTA 16 1            AAGAGT TTppGGpp TAppGpATTpApppT 17 1ApAGTpppApAGTTTAA TTppGGpp GTAGppGpT 18 1      ATAppAGGGTpG TTppGGppAAGpGpTGTT 19 1               GAG TTppGGpp TAGpGpAGAAGpppTGGAT 20 1      TTTTppAGGAA TTppGGpp AAGpGpTGTGA 21 1     AATTAppTGAGGA TTppGGppAAGpGpAGA 22 1      pTGpGTTApGpp TTppGGpp TGGpTGATAG 23 1  TAppTGAGTTATGTA TTppGGpp GTGpGpA 24 1           pAAAGpA TTppGGppTTGpGpAGTAGpppT 25 1   GTAGTTppAGATTGA TTppGGpp TTGpGpT 26 1  AATApTppAGGTGAG TTppGGpp AAGpGpT

PCSK9-binding aptamers of the conserved sequence family from pool 11730(Nap-dC/Tyr-dU) are shown in Table 16. Only the random region of eachsequence is shown. The number of copies of each sequence (identical orequivalent with up to 5 mismatches) out of 17,695 total sequences isindicated. All aptamers in this family share the conserved sequenceelement yGpppG, where p=Nap-dC and Y=Tyr-dU. Many sequences alsocontained the conserved sequence element yyAyGpAp. Aptamer 11730-19 (SEQID No: 27, K_(d)=0.2 nM) was the representative chosen from this poolfor the metabolic stability assay.

TABLE 16 Aptamers from pool 11730 SEQ ID No CopiesRandom Region Sequence 27 680    GyyAypGpAAyGyGpGpppGGG yGpppG pp 28 163                       yG yGpppG GAyAyyAApyGyyppGAGpAGy 29 56             yGyyyAyGpApA yGpppG pGAyGApAGyAA 30 44         AGyGyGAyyAyGpApy yGpppG pAyyyGGy 31 15       yAyAGAApAyAAyGpApA yGpppG pAyApy 32 15          yAypAGyyyAyGpApGyGpppG pGAyGApy 33 13     GApyApGAGGGAyGAyGpApA yGpppG pAy 34 11         AyAAyGAyyAyGpApA yGpppG pAyGypAy 35 4                 GGpAypGyGyGpppG AyyyypyAAppGGGA 36 4          GppGAAyyyAyGpApp yGpppG pAyGAyyp 372            ppAAypAyGApApA yGpppG GAyGAyApy 38 1                    yApGA yGpppG GAyAyyGApyGyyppGypG 39 1      pGyAGpGApGGGpGyGGpA yGpppG Gppppp 40 1                 yGGyGAGAGyGpppG GAyAyyAApyGyypp 41 1              ypAAAGGppGyG yGpppGAyyyypyAAppG 42 1 yyypGAAGyyGAGpGyGGpAAyApy yGppp 43 1           pGyGyyyAyGpApy yGpppG pGAyyApApp

PCSK9-binding aptamers of the conserved sequence family from pool 11733(Pp-dC/Nap-dU) are shown in Table 17. Only the random region of eachsequence is shown. The number of copies of each sequence (identical orequivalent with up to 5 mismatches) out of 16,118 total sequences isindicated. All aptamers in this family share the conserved sequenceelement rPPPAAGGrrPAPPG (SEQ ID NO: 83), where r=Pp-dC and P=Nap-dU.Aptamer 11733-44 (SEQ ID NO: 44, SL1063) was the most potent 30-merinhibitor of wild-type human PCSK9 (IC₅₀=2.8 nM). Aptamer 11733-198 (SEQID No: 46, K_(d)=0.07 nM) was the representative chosen from this poolfor the metabolic stability assay.

TABLE 17 aptamers from pool 11733 SEQ ID No CopiesRandom Region Sequence 44 1041           AArGpA rpppAAGGrrpAppGAGGAAArpr 45 969               rA rpppAAGArrpAppG rGGAGArrrpGGG 46 340            rGpG rpppAAGArrpGppG AGApGrGrprA 47 204                ArpppAAGArrpGppG AGGGrrprGGGAAp 48 158           GrrGGp GpppAAGAArpGppGGGGrArppr 49 154            GrGrA rpppAAGArrpAppG GGGAGAArpr 50 127 pppAAGGrrpGppG AGGArrprGGrApGAA 51 105              rAA rpppAAGGrrpGppGGGAGAGrrrppG 52 51       GAApArrArG rpppAAGArrpAppG GAprG 53 23           GAGAA rpppAAGArrpAppG AGGGrrprpG 54 13              rGArpppAAGGrrpAppG GGGGArprGAr 55 11            GAArA rpppAAGArrpGppGGGAAGrGprp 56 10                A rpppAAGGrrpGppG AGGAAArrGprpGA 57 10              AG rpppAAGArrpGppG AGAApprGArAAA 58 8               AGrpppAAGArrpGppG AGrAGrrprGArr 59 7  GAAGGprAAGpGGrA rpppAAGGrrpGppr 60 7          AAArrA rpppAAGArrpGppG GGGrArprp 61 6             AAArrpppAAGArrpAppG AGGpGGGrprA 62 6           pAGGrG rpppAAGArrpAppGAGGGArprr 63 4           GpGArG rpppAAGArrpAppA GGGrArppr 64 3         GAAArrA rpppAAGArrpAppG AGAGArpr 65 3             GAAArpppAAGArrpAppG AGrAGGAArpr 67 3              rGA rpppAAGArrpGppGAAAGprGrGGGG 68 3                A rpppAAGArrpGppG AGArrGGrprGGArAp 69 2             GAA rpppAAGArrpGppG AGGArrAprArG 70 2  ArGpGGAGprGGArArpppAAGGrrpGppr 71 2           prGrGA rpppAAGGrrpAppG pGGGArprp 72 2               G rpppAAGGrrpAppG AGAprGGrprGGGp 73 2               GArpppAAGGrrpGppG AAGGprGAGAGpp 74 2           AArGAp rpppAAGGrrpGppAGGGGrppr 75 2   rAAGrpGrrprGrA rpppAAGArGpGppG G 76 2            ArrGpGpppAAGAArpGppG GGGGGAArpr 77 1       GAApArrArG rpppAAGArrpAppGAGApGrGrprA 78 1 pAAGrrpGAGAAArrA rpppAAGArrpppp 79 1             rGAArpppAAGArrpGppG AGrAGrprppr 80 1            AGAAp rpppAAGGrrpGprGAGArrprGAG

1. An aptamer comprising at least one first 5-position modifiedpyrimidine and at least one second 5-position modified pyrimidine,wherein the first 5-position modified pyrimidine and the second5-position modified pyrimidine are different 5-position modifiedpyrimidines; wherein the first 5-position modified pyrimidine is a5-position modified uridine and wherein the second 5-position modifiedpyrimidine is a 5-position modified cytidine; or wherein the first5-position modified pyrimidine is a 5-position modified cytidine andwherein the second 5-position modified pyrimidine is a 5-positionmodified uridine; wherein the 5-position modified cytidine is PPdC andthe 5-position modified uridine is TyrdU; or wherein the 5-positionmodified cytidine is PPdC and the 5-position modified uridine is PPdU;or wherein the 5-position modified cytidine is PPdC and the 5-positionmodified uridine is NapdU; or wherein the 5-position modified cytidineis PPdC and the 5-position modified uridine is ThrdU; or wherein the5-position modified cytidine is NapdC and the 5-position modifieduridine is NapdU; or wherein the 5-position modified cytidine is NapdCand the 5-position modified uridine is PPdU; or wherein the 5-positionmodified cytidine is NapdC and the 5-position modified uridine is ThrdU;or wherein the 5-position modified cytidine is NapdC and the 5-positionmodified uridine is MOEdU.
 2. The aptamer of claim 1, wherein the moietyof the 5-position modified uridine is covalently linked via a linkercomprising a group selected from an amide linker, a carbonyl linker, apropynyl linker, an alkyne linker, an ester linker, a urea linker, acarbamate linker, a guanidine linker, an amidine linker, a sulfoxidelinker, and a sulfone linker.
 3. The aptamer of claim 1, wherein themoiety of the 5-position modified cytidine is covalently linked via alinker comprising a group selected from an amide linker, a carbonyllinker, a propynyl linker, an alkyne linker, an ester linker, a urealinker, a carbamate linker, a guanidine linker, an amidine linker, asulfoxide linker, and a sulfone linker.
 4. (canceled)
 5. (canceled) 6.(canceled)
 7. (canceled)
 8. (canceled)
 9. (canceled)
 10. (canceled) 11.The aptamer of claim 1, wherein the aptamer is 20 to 100, or 20 to 90,or 20 to 80, or 20 to 70, or 20 to 60, or 20 to 50, or 30 to 100, or 30to 90, or 30 to 80, or 30 to 70, or 30 to 60, or 30 to 50, or 40 to 100,or 40 to 90, or 40 to 80, or 40 to 70, or 40 to 60, or 40 to 50nucleotides in length.
 12. A composition comprising a plurality ofpolynucleotides, wherein each polynucleotide comprises at least onefirst 5-position modified pyrimidine and at least one second 5-positionmodified pyrimidine, wherein the first 5-position modified pyrimidineand the second 5-position modified pyrimidine are different 5-positionmodified pyrimidines; wherein the first 5-position modified pyrimidineis a 5-position modified uridine and wherein the second 5-positionmodified pyrimidine is a 5-position modified cytidine; or wherein thefirst 5-position modified pyrimidine is a 5-position modified cytidineand wherein the second 5-position modified pyrimidine is a 5-positionmodified uridine; wherein the 5-position modified cytidine is PPdC andthe 5-position modified uridine is TyrdU; or wherein the 5-positionmodified cytidine is PPdC and the 5-position modified uridine is PPdU;or wherein the 5-position modified cytidine is PPdC and the 5-positionmodified uridine is NapdU; or wherein the 5-position modified cytidineis PPdC and the 5-position modified uridine is ThrdU; or wherein the5-position modified cytidine is NapdC and the 5-position modifieduridine is NapdU; or wherein the 5-position modified cytidine is NapdCand the 5-position modified uridine is PPdU; or wherein the 5-positionmodified cytidine is NapdC and the 5-position modified uridine is ThrdU;or wherein the 5-position modified cytidine is NapdC and the 5-positionmodified uridine is MOEdU.
 13. The composition of claim 12, wherein eachpolynucleotide comprises a fixed region at the 5′ end of thepolynucleotide and/or the 3′ end of the polynucleotide.
 14. Thecomposition of claim 13, wherein the fixed region at the 5′ end of eachpolynucleotide is at least 10, at least 15, at least 20, at least 25 orat least 30 nucleotides in length, or 5 to 30, 10 to 30, 15 to 30, 5 to20, or 10 to 20 nucleotides in length.
 15. (canceled)
 16. Thecomposition of claim 13, wherein the fixed region at the 3′ end of thepolynucleotide is at least 10, at least 15, at least 20, at least 25 orat least 30 nucleotides in length, or 5 to 30, 10 to 30, 15 to 30, 5 to20, or 10 to 20 nucleotides in length.
 17. (canceled)
 18. (canceled) 19.(canceled)
 20. (canceled)
 21. (canceled)
 22. (canceled)
 23. (canceled)24. The composition of claim 12, wherein each polynucleotide comprises arandom region.
 25. (canceled)
 26. The composition of claim 12, whereineach polynucleotide is 20 to 100, or 20 to 90, or 20 to 80, or 20 to 70,or 20 to 60, or 20 to 50, or 30 to 100, or 30 to 90, or 30 to 80, or 30to 70, or 30 to 60, or 30 to 50, or 40 to 100, or 40 to 90, or 40 to 80,or 40 to 70, or 40 to 60, or 40 to 50 nucleotides in length.
 27. Acomposition comprising an aptamer and a target, wherein the aptamer andthe target are capable of forming a complex, and wherein the aptamer isan aptamer of claim
 1. 28. A composition comprising a first aptamer, asecond aptamer, and a target, wherein the first aptamer comprises atleast one first 5-position modified pyrimidine and at least one second5-position modified pyrimidine; wherein the second aptamer comprises atleast one third 5-position modified pyrimidine or wherein the secondaptamer comprises at least one third 5-position modified pyrimidine andat least one fourth 5-position modified pyrimidine; wherein the firstaptamer, second aptamer and the target are capable of forming a trimercomplex; and wherein the first 5-position modified pyrimidine and thesecond 5-position modified pyrimidine are different 5-position modifiedpyrimidines; and wherein the first aptamer is an aptamer of claim
 1. 29.The composition of claim 28, wherein the target is selected from aprotein, a peptide, a carbohydrate, a small molecule, a cell and atissue.
 30. A method comprising: (a) contacting an aptamer capable ofbinding to a target molecule with a sample; (b) incubating the aptamerwith the sample to allow an aptamer-target complex to form; (c)enriching for the aptamer-target complex in the sample and (d) detectingfor the presence of the aptamer, aptamer-target complex or targetmolecule, wherein the detection of the aptamer, aptamer-target complexor target molecule indicates that the target molecule is present in thesample, and wherein the lack of detection of the aptamer, aptamer-targetcomplex or target molecule indicates that the target molecule is notpresent in the sample; wherein the aptamer is an aptamer of claim
 1. 31.(canceled)
 32. (canceled)
 33. (canceled)
 34. (canceled)
 35. (canceled)36. The method of claim 30, wherein the sample is selected from wholeblood, leukocytes, peripheral blood mononuclear cells, plasma, serum,sputum, breath, urine, semen, saliva, meningial fluid, amniotic fluid,glandular fluid, lymph fluid, nipple aspirate, bronchial aspirate,synovial fluid, joint aspirate, cells, a cellular extract, stool,tissue, a tissue biopsy, and cerebrospinal fluid.
 37. A method fordetecting a target in a sample comprising a) contacting the sample witha first aptamer to form a mixture, wherein the first aptamer is capableof binding to the target to form a first complex; b) incubating themixture under conditions that allow for the first complex to form; c)contacting the mixture with a second aptamer, wherein the second aptameris capable of binding the first complex to form a second complex; d)incubating the mixture under conditions that allow for the secondcomplex to form; e) detecting for the presence or absence of the firstaptamer, the second aptamer, the target, the first complex or the secondcomplex in the mixture, wherein the presence of the first aptamer, thesecond aptamer, the target, the first complex or the second complexindicates that the target is present in the sample; wherein the firstaptamer is an aptamer of claim 1; wherein the second aptamer comprisesat least one third 5-position modified pyrimidine, or wherein the secondaptamer comprises at least one third 5-position modified pyrimidine andat least one fourth 5-position modified pyrimidine.
 38. (canceled) 39.The method of claim 37, wherein the first aptamer, second aptamer andthe target are capable of forming a trimer complex.
 40. A method foridentifying one or more aptamers capable of binding to a target moleculecomprising: (a) contacting a library of aptamers with the targetmolecule to form a mixture, and allowing for the formation of anaptamer-target complex, wherein the aptamer-target complex forms when anaptamer has affinity for the target molecule; (b) partitioning theaptamer-target complex from the remainder of the mixture (or enrichingfor the aptamer-target complex); (c) dissociating the aptamer-targetcomplex; and (d) identifying the one or more aptamers capable of bindingto the target molecule; wherein the library of aptamers comprises aplurality of polynucleotides, wherein each polynucleotide comprises atleast one first 5-position modified pyrimidine and at least one second5-position modified pyrimidine, wherein the first 5-position modifiedpyrimidine and the second 5-position modified pyrimidine are different5-position modified pyrimidines; wherein the first 5-position modifiedpyrimidine is a 5-position modified uridine and wherein the second5-position modified pyrimidine is a 5-position modified cytidine; orwherein the first 5-position modified pyrimidine is a 5-positionmodified cytidine and wherein the second 5-position modified pyrimidineis a 5-position modified uridine; wherein the 5-position modifiedcytidine is PPdC and the 5-position modified uridine is TyrdU; orwherein the 5-position modified cytidine is PPdC and the 5-positionmodified uridine is PPdU; or wherein the 5-position modified cytidine isPPdC and the 5-position modified uridine is NapdU; or wherein the5-position modified cytidine is PPdC and the 5-position modified uridineis ThrdU; or wherein the 5-position modified cytidine is NapdC and the5-position modified uridine is NapdU; or wherein the 5-position modifiedcytidine is NapdC and the 5-position modified uridine is PPdU; orwherein the 5-position modified cytidine is NapdC and the 5-positionmodified uridine is ThrdU; or wherein the 5-position modified cytidineis NapdC and the 5-position modified uridine is MOEdU.
 41. (canceled)42. (canceled)
 43. (canceled)
 44. (canceled)
 45. (canceled) 46.(canceled)
 47. (canceled)
 48. (canceled)
 49. (canceled)
 50. (canceled)51. (canceled)
 52. (canceled)
 53. (canceled)
 54. (canceled) 55.(canceled)
 56. (canceled)
 57. (canceled)
 58. (canceled)
 59. (canceled)60. (canceled)
 61. (canceled)
 62. The aptamer of claim 1, wherein theaptamer has improved nuclease stability and/or a longer half-life inhuman serum compared to an aptamer of the same length and nucleobasesequence that comprises an unmodified pyrimidine in place of each of thefirst 5-position modified pyrimidines or an unmodified pyrimidine inplace of each of the second 5-position modified pyrimidine.